When, where and which PIK3CA mutations are pathogenic in congenital disorders

Altres ajuts: PTEN RESEARCH Foundation (BRR-17-001/IJC-21-001); Fundació La Caixa (LCF/PR/PR16/51110035 i LCF/PR/HR19/52160023)PIK3CA encodes the class I PI3Kα isoform and is frequently mutated in cancer. Activating mutations in PIK3CA also cause a range of congenital disorders featuring asymmetric tissue overgrowth, known as the PIK3CA-related overgrowth spectrum (PROS), with frequent vascular involvement. In PROS, PIK3CA mutations arise postzygotically, during embryonic development, leading to a mosaic body pattern distribution resulting in a variety of phenotypic features. A clear skewed pattern of overgrowth favoring some mesoderm-derived and ectoderm-derived tissues is observed but not understood. Here, we summarize our current knowledge of the determinants of PIK3CA-related pathogenesis in PROS, including intrinsic factors such as cell lineage susceptibility and PIK3CA variant bias, and extrinsic factors, which refers to environmental modifiers. We also include a section on PIK3CA-related vascular malformations given that the vasculature is frequently affected in PROS. Increasing our biological understanding of PIK3CA mutations in PROS will contribute toward unraveling the onset and progression of these conditions and ultimately impact on their treatment. Given that PIK3CA mutations are similar in PROS and cancer, deeper insights into one will also inform about the other. In this Review, the authors provide an overview of the pathogenic effects of somatic activating PIK3CA mutations in congenital disorders and discuss how the interplay between genetics, cell identity and the environment explains the onset, progression and severity of these disorders with a special focus on the vasculature

A junctional PACSIN2/EHD4/MICAL-L1 complex coordinates VE-cadherin trafficking for endothelial migration and angiogenesis

Angiogenic sprouting relies on collective migration and coordinated rearrangements of endothelial leader and follower cells. VE-cadherin-based adherens junctions have emerged as key cell-cell contacts that transmit forces between cells and trigger signals during collective cell migration in angiogenesis. However, the underlying molecular mechanisms that govern these processes and their functional importance for vascular development still remain unknown. We previously showed that the F-BAR protein PACSIN2 is recruited to tensile asymmetric adherens junctions between leader and follower cells. Here we report that PACSIN2 mediates the formation of endothelial sprouts during angiogenesis by coordinating collective migration. We show that PACSIN2 recruits the trafficking regulators EHD4 and MICAL-L1 to the rear end of asymmetric adherens junctions to form a recycling endosome-like tubular structure. The junctional PACSIN2/EHD4/MICAL-L1 complex controls local VE-cadherin trafficking and thereby coordinates polarized endothelial migration and angiogenesis. Our findings reveal a molecular event at force-dependent asymmetric adherens junctions that occurs during the tug-of-war between endothelial leader and follower cells, and allows for junction-based guidance during collective migration in angiogenesis

Therapeutic benefit of selective inhibition of p110α PI3-kinase in pancreatic neuroendocrine tumors

Purpose: mutations in the PI3K pathway occur in 16% of patients with pancreatic neuroendocrine tumors (PanNETs), which suggests that these tumors are an exciting setting for PI3K/AKT/mTOR pharmacologic intervention. Everolimus, an mTOR inhibitor, is being used to treat patients with advanced PanNETs. However, resistance tomTOR-targeted therapy is emerging partially due to the loss of mTOR-dependent feedback inhibition of AKT. In contrast, the response to PI3K inhibitors in PanNETs is unknown. Experimental Design: in the current study, we assessed the frequency of PI3K pathway activation in human PanNETs and in RIP1-Tag2 mice, a preclinical tumor model of PanNETs, and we investigated the therapeutic efficacy of inhibiting PI3K in RIP1-Tag2 mice using a combination of pan (GDC-0941) and p110 alpha-selective (GDC-0326) inhibitors and isoform-specific PI3K kinase-dead-mutant mice. Results: human and mouse PanNETs showed enhanced pAKT, pPRAS40, and pS6 positivity compared with normal tissue. Although treatment of RIP1-Tag2 mice with GDC-0941 led to reduced tumor growth with no impact on tumor vessels, the selective inactivation of the p110 alpha PI3K isoform, either genetically or pharmacologically, reduced tumor growth as well as vascular area. Furthermore, GDC-0326 reduced the incidence of liver and lymph node metastasis compared with vehicle-treated mice. We also demonstrated that tumor and stromal cells are implicated in the antitumor activity of GDC-0326 in RIP1-Tag2 tumors. Conclusions: our data provide a rationale for p110a-selective intervention in PanNETs and unravel a new function of this kinase in cancer biology through its role in promoting metastasis

PI3K-C2β limits mTORC1 signaling and angiogenic growth

Phosphoinositide 3-kinases (PI3Ks) phosphorylate intracellular inositol lipids to regulate signaling and intracellular vesicular trafficking. Mammals have eight PI3K isoforms, of which class I PI3Kα and class II PI3K-C2α are essential for vascular development. The class II PI3K-C2β is also abundant in endothelial cells. Using in vivo and in vitro approaches, we found that PI3K-C2β was a critical regulator of blood vessel growth by restricting endothelial mTORC1 signaling. Mice expressing a kinase-inactive form of PI3K-C2β displayed enlarged blood vessels without corresponding changes in endothelial cell proliferation or migration. Instead, inactivation of PI3K-C2β resulted in an increase in the size of endothelial cells, particularly in the sprouting zone of angiogenesis. Mechanistically, we showed that the aberrantly large size of PI3K-C2β mutant endothelial cells was caused by mTORC1 activation, which sustained growth in these cells. Consistently, pharmacological inhibition of mTORC1 with rapamycin normalized vascular morphogenesis in PI3K-C2β mutant mice. Together, these results identify PI3K-C2β as a crucial determinant of endothelial signaling and illustrate the importance of mTORC1 regulation during angiogenic growth

Somatic activating mutations in Pik3ca cause sporadic venous malformations in mice and humans.

Venous malformations (VMs) are painful and deforming vascular lesions composed of dilated vascular channels, which are present from birth. Mutations in the TEK gene, encoding the tyrosine kinase receptor TIE2, are found in about half of sporadic (nonfamilial) VMs, and the causes of the remaining cases are unknown. Sclerotherapy, widely accepted as first-line treatment, is not fully efficient, and targeted therapy for this disease remains underexplored. We have generated a mouse model that faithfully mirrors human VM through mosaic expression of Pik3ca(H1047R), a constitutively active mutant of the p110α isoform of phosphatidylinositol 3-kinase (PI3K), in the embryonic mesoderm. Endothelial expression of Pik3ca(H1047R)resulted in endothelial cell (EC) hyperproliferation, reduction in pericyte coverage of blood vessels, and decreased expression of arteriovenous specification markers. PI3K pathway inhibition with rapamycin normalized EC hyperproliferation and pericyte coverage in postnatal retinas and stimulated VM regression in vivo. In line with the mouse data, we also report the presence of activating PIK3CA mutations in human VMs, mutually exclusive with TEK mutations. Our data demonstrate a causal relationship between activating Pik3ca mutations and the genesis of VMs, provide a genetic model that faithfully mirrors the normal etiology and development of this human disease, and establish the basis for the use of PI3K-targeted therapies in VMs.Postdoctoral fellowships were from EMBO (A LTF 165-2013) to S.D.C, EU Marie Curie (MEIF-CT-2005-010264) to E.T. and EU Marie Curie (PIIF-GA-2009-252846) to I.M.B. M.Z.-T. is supported by the EPSRC Early Career Fellowship of T.L.K. (EP/L006472/1). D.J.S. is a BHF Intermediate Basic Science Research Fellow (FS/15/33/31608). A.L.D is supported by the UK NIHR Joint UCL/University College London Hospitals Biomedical Research Centre. V.E.R.P. was supported by the Wellcome Trust (097721/Z/11/Z). R.K.S. is supported by the Wellcome Trust (WT098498), the Medical Research Council (M RC_MC_UU_12012/5). R.G.K. is supported by the NIHR Rare Diseases Translational Research Collaboration. V.W. is supported by the European FPVI Integrated Project ‘Eurostemcell’. M.F.L. and A.B. are supported by the King’s College London and UCL Comprehensive Cancer Imaging Centre CR-UK and EPSRC, in association with the MRC and DoH (England). W.A.P. is supported by funding from the National Health and Medical Research Council (NHMRC) of Australia. Work in the laboratory of M.G. is supported by research grants SAF2013-46542-P and SAF2014-59950-P from MICINN (Spain), 2014-SGR-725 from the Catalan Government, the People Programme (Marie Curie Actions) from the European Union's Seventh Framework Programme FP7/2007-2013/ (REA grant agreement 317250), the Institute of Health Carlos III (ISC III) and the European Regional Development Fund (ERDF) under the integrated Project of Excellence no. PIE13/00022 (ONCOPROFILE). Work in the laboratory of B.V. is supported by Cancer Research UK (C23338/A15965) and the UK NIHR University College London Hospitals Biomedical Research Centre.This is the author accepted manuscript. The final version is available from the American Association for the Advancement of Science via http://dx.doi.org/10.1126/scitranslmed.aad998

Regulation of actomyosin contractility by p110α P13-kinase in sprouting angiogenesis

[eng] Class IA PI3K (PI3K) functions have been widely investigated over the last two decades. PI3K signalling is located at the crossroads of many cell surface receptors sending signals to coordinate multiple cellular functions such as cell growth, survival, motility, and metabolism. Fine-tune regulation of PI3K signalling in cells is needed to ensure the functionality of tissues and organs. However, it is still not clear how or even which PI3K isoforms are concerted into precise morphogenic events. On the other hand, PI3K activity plays central roles in several cellular processes critical for cancer progression. Hence, PI3K pathway inhibition is considered an important target for therapeutic intervention in cancer, and progress in the clinical area is being monitored by many clinical trials with PI3K inhibitors. We were interested in investigating the role of PI3K activity in endothelial cells during the process of angiogenesis. Although ECs express all class I PI3K isoforms, only inactivation of the catalytic subunit p110α in endothelial cells (not p110β or p110δ inactivation) leads to vascular defects in the embryo (Graupera et al. 2008). This indicates that p110α activity in ECs is required in a cell- autonomous manner to ensure proper vascular development and remodelling during the embryogenesis. However, progress in the understanding of how p110α-PI3K signalling regulates the different steps of vascular morphogenesis has been hampered by embryonic lethality that both the constitutive and endothelial specific p110α mutant mice exhibit. By using a tamoxifen-inducible endothelial Cre line in mouse and genetic and pharmacological approaches in zebrafish embryos, we have found that p110α signalling is required to maintain vessel stability. The lack of p110α activity leads to endothelial tubular structures composed of single cells that show an elongated shape with multiple protrusions and no lumen. These ECs fail in the elongation of the inter-endothelial contacts during the sprout outgrowth. Furthermore, I found that p110α is involve in the initial steps of fusion and is necessary for proper establishment of a new connection. Finally, I identifyed that p110α negatively controls actomyosin contractility independently of Rho/ROCK signalling pathway and that this control could be exerted through the regulation of MLC phosphatase activity by the impact on mRIP and/or MYPT proteins.[spa] La señalización PI3K de clase IA se requiere de una manera autónoma en células endoteliales para el correcto crecimiento de los vasos sanguíneos. Aunque las células endoteliales expresan todas las isoformas de la clase IA de PI3Ks, sólo la subunidad catalítica p110α es necesaria para la angiogenesis fisiológica. Sin embargo, poco se sabe sobre el papel de p110α -PI3K en las diferentes etapas de la morfogénesis vascular. Mediante la generación de una línea inducible Cre endotelial de ratón y la inactivación genética y farmacológica de la proteína en embriones de pez cebra, hemos encontrado que la señalización a través de la proteína p110α es necesaria para mantener la estabilidad del los vasos sanguíneos. La falta de la actividad de la proteína p110α da lugar a la formación de una vasculatura aberrante formada por estructuras endoteliales muy delgadas compuestas por células individuales que emiten múltiples protrusiones y carecen de lumen. Durante la elongación del nuevo brote vascular las células endoteliales no puede elongar la superficie de adhesión entre células endoteliales y por tanto no pueden sufrir reordenamientos necesarios para el crecimiento del nuevo vaso. También hemos visto que la proteína p110α está implicada en la estabilización de los nuevos contactos durante el proceso de anastomosis vascular y su inactivación da lugar a inestabilidad vascular y la aparición de desconexiones en entre vasos. La falta de la proteína p110α se asocia con un aumento en la formación de los cables de actina cortical e hiperfosforilacion de la cadena ligera de la miosina. Por tanto, identificamos que la ruta de señalización p110α-PI3K controla negativamente la contractilidad de las fibras de actomiosina de forma independiente a la via de señalización Rho-ROCK y que este control podría ser ejercido a través de la regulación de la actividad de la fosfatasa MLC a través de las proteínas mRIP y/o MYPT1

Endothelial cell rearrangements during vascular patterning require PI3-kinase-mediated inhibition of actomyosin contractility

Angiogenesis is a dynamic process relying on endothelial cell rearrangements within vascular tubes, yet the underlying mechanisms and functional relevance are poorly understood. Here we show that PI3Kα regulates endothelial cell rearrangements using a combination of a PI3Kα-selective inhibitor and endothelial-specific genetic deletion to abrogate PI3Kα activity during vessel development. Quantitative phosphoproteomics together with detailed cell biology analyses in vivo and in vitro reveal that PI3K signalling prevents NUAK1-dependent phosphorylation of the myosin phosphatase targeting-1 (MYPT1) protein, thereby allowing myosin light chain phosphatase (MLCP) activity and ultimately downregulating actomyosin contractility. Decreased PI3K activity enhances actomyosin contractility and impairs junctional remodelling and stabilization. This leads to overstretched endothelial cells that fail to anastomose properly and form aberrant superimposed layers within the vasculature. Our findings define the PI3K/NUAK1/MYPT1/MLCP axis as a critical pathway to regulate actomyosin contractility in endothelial cells, supporting vascular patterning and expansion through the control of cell rearrangement.CERCA Programme/Generalitat de Catalunya for institutional support. This work was funded by Ministerio de Ciencia, Innovación y Universidades, which is part of Agencia Estatal de Investigación (AEI, Spain) through the projects SAF2014-59950-P, SAF2017-82072-ERC, and SAF2017-89116-R co-funded by European Regional Developmental Fund (ERDF), a way to build Europe; by the Catalan Government through the projects 2014-SGR and 2017-SGR; and by la Fundació Bancària “La CaixaPeer Reviewe

Cell-cell junctions as sensors and transducers of mechanical forces

Epithelial and endothelial monolayers are multicellular sheets that form barriers between the ‘outside’ and ‘inside’ of tissues. Cell-cell junctions, made by adherens junctions, tight junctions and desmosomes, hold together these monolayers. They form intercellular contacts by binding their receptor counterparts on neighboring cells and anchoring these structures intracellularly to the cytoskeleton. During tissue development, maintenance and pathogenesis, monolayers encounter a range of mechanical forces from the cells themselves and from external systemic forces, such as blood pressure or tissue stiffness. The molecular landscape of cell-cell junctions is diverse, containing transmembrane proteins that form intercellular bonds and a variety of cytoplasmic proteins that remodel the junctional connection to the cytoskeleton. Many junction-associated proteins participate in mechanotransduction cascades to confer mechanical cues into cellular responses that allow monolayers to maintain their structural integrity. We will discuss force-dependent junctional molecular events and their role in cell-cell contact organization and remodeling

Phosphoinositide 3-Kinase-Regulated pericyte maturation governs vascular remodeling

© 2020 American Heart Association, Inc.Background: Pericytes regulate vessel stabilization and function, and their loss is associated with diseases such as diabetic retinopathy or cancer. Despite their physiological importance, pericyte function and molecular regulation during angiogenesis remain poorly understood. Methods: To decipher the transcriptomic programs of pericytes during angiogenesis, we crossed Pdgfrb(BAC)-CreERT2 mice into RiboTagflox/flox mice. Pericyte morphological changes were assessed in mural cell-specific R26-mTmG reporter mice, in which low doses of tamoxifen allowed labeling of single-cell pericytes at high resolution. To study the role of phosphoinositide 3-kinase (PI3K) signaling in pericyte biology during angiogenesis, we used genetic mouse models that allow selective inactivation of PI3Kα and PI3Kβ isoforms and their negative regulator phosphate and tensin homolog deleted on chromosome 10 (PTEN) in mural cells. Results: At the onset of angiogenesis, pericytes exhibit molecular traits of cell proliferation and activated PI3K signaling, whereas during vascular remodeling, pericytes upregulate genes involved in mature pericyte cell function, together with a remarkable decrease in PI3K signaling. Immature pericytes showed stellate shape and high proliferation, and mature pericytes were quiescent and elongated. Unexpectedly, we demonstrate that PI3Kβ, but not PI3Kα, regulates pericyte proliferation and maturation during vessel formation. Genetic PI3Kβ inactivation in pericytes triggered early pericyte maturation. Conversely, unleashing PI3K signaling by means of PTEN deletion delayed pericyte maturation. Pericyte maturation was necessary to undergo vessel remodeling during angiogenesis. Conclusions: Our results identify new molecular and morphological traits associated with pericyte maturation and uncover PI3Kβ activity as a checkpoint to ensure appropriate vessel formation. In turn, our results may open new therapeutic opportunities to regulate angiogenesis in pathological processes through the manipulation of pericyte PI3Kβ activity.Dr Graupera’s laboratory is supported by the research grants SAF2017-89116R-P from Ministerio de Ciencia (Spain) cofunded by European Regional Developmental Fund (ERDF), a Way to Build Europe; by the Catalan government through the project 2017-SGR; by La Caixa Foundation (HR18-00120); by la Asociación Española contra el Cancer (AECC)-Grupos Traslacionales (GCTRA18006CARR); by la Fundación BBVA (Beca Leonardo a Investigadores y Creadores Culturales 2017); and by the People Program (Marie Curie Actions; grant agreement 317250) of the European Union’s Seventh Framework Program FP7/2007 to 2013/, and the Marie Skłodowska-Curie (grant agreement 675392) of the European Union’s Horizon 2020 research. Dr Carracedo’s laboratory is supported by the Basque Department of Industry, Tourism and Trade (Elkartek) and the Department of Education (IKERTALDE IT1106-16), the Ministerio de Ciencia (SAF2016-79381-R [FEDER/EU], Severo Ochoa Excellence Accreditation SEV-2016-0644; Excellence Networks SAF2016-81975-REDT), European Training Networks Project (H2020-MSCA-ITN-308 2016 721532), the AECC (IDEAS175CARR, GCTRA18006CARR), La Caixa Foundation (HR17-00094), and the European Research Council (StG 336343, PoC 754627, CoG 819242). Centro de Investigación Biomédica en Red Cáncer (CIBERONC) was cofunded with FEDER funds and funded by Instituto de Salud Carlos III. Dr Aransay’s laboratory is supported by the Basque Department of Industry, Tourism and Trade (Elkartek) and the Severo Ochoa Excellence Accreditation SEV-2016-0644. Dr Franco was supported by European Research Council (StG 679368), the H2020-Twinning grant (692322), the Fundação para a Ciência e a Tecnologia funding (grants IF/00412/2012; EXPL-BEX-BCM-2258-2013; PRECISE-LISBOA-01-0145-FEDER-016394), and a grant from the Fondation Leducq (17CVD03). Personal support was from Marie-Curie ITN Actions (Dr Figueiredo and Kobialka), Juan de la Cierva (IJCI-2015-23455, Dr Villacampa), and CIBERONC (A. Martinez-Romero).info:eu-repo/semantics/publishedVersio

Therapeutic benefit of selective inhibition of p110α PI3-kinase in pancreatic neuroendocrine tumors

Purpose: mutations in the PI3K pathway occur in 16% of patients with pancreatic neuroendocrine tumors (PanNETs), which suggests that these tumors are an exciting setting for PI3K/AKT/mTOR pharmacologic intervention. Everolimus, an mTOR inhibitor, is being used to treat patients with advanced PanNETs. However, resistance tomTOR-targeted therapy is emerging partially due to the loss of mTOR-dependent feedback inhibition of AKT. In contrast, the response to PI3K inhibitors in PanNETs is unknown. Experimental Design: in the current study, we assessed the frequency of PI3K pathway activation in human PanNETs and in RIP1-Tag2 mice, a preclinical tumor model of PanNETs, and we investigated the therapeutic efficacy of inhibiting PI3K in RIP1-Tag2 mice using a combination of pan (GDC-0941) and p110 alpha-selective (GDC-0326) inhibitors and isoform-specific PI3K kinase-dead-mutant mice. Results: human and mouse PanNETs showed enhanced pAKT, pPRAS40, and pS6 positivity compared with normal tissue. Although treatment of RIP1-Tag2 mice with GDC-0941 led to reduced tumor growth with no impact on tumor vessels, the selective inactivation of the p110 alpha PI3K isoform, either genetically or pharmacologically, reduced tumor growth as well as vascular area. Furthermore, GDC-0326 reduced the incidence of liver and lymph node metastasis compared with vehicle-treated mice. We also demonstrated that tumor and stromal cells are implicated in the antitumor activity of GDC-0326 in RIP1-Tag2 tumors. Conclusions: our data provide a rationale for p110a-selective intervention in PanNETs and unravel a new function of this kinase in cancer biology through its role in promoting metastasis