An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Aix Marseille University

KU Leuven Campus Gasthuisberg

Integrins in invasive growth.

PINCH is a five LIM domain protein involved in the regulation of integrin-mediated cell adhesion. It has been shown that PINCH interacts with integrin-linked kinase and Nck2. Here we describe a new isoform of PINCH, which we call PINCH2. Therefore, we rename PINCH to PINCH1. PINCH2 has an overall similarity of 92% to PINCH1 and contains five LIM domains like PINCH1. While protein and gene structure of the PINCH homologues are very similar and well conserved during evolution, we observed differential expression pattern of the mRNAs. Based on northern hybridization of mouse embryo RNA, PINCH1 is already detectable at E8.5. It is highly expressed during later stages of development and in all adult mouse tissues analyzed, with the highest levels in heart, lung, bladder, skin, and uterus. In contrast, significant PINCH2 expression starts at E14.5. In adult mice it is widely expressed, similar to PINCH1, but absent from spleen and thymus. In situ hybridization confirmed the Northern data and showed differential expression of PINCH1 and PINCH2 in embryonic intestine. Finally, we demonstrate that PINCH2 localizes to focal adhesions in NIH 3T3 cells and to Z-disks in primary rat cardiomyocytes.

PINCH2 is a new five LIM domain protein, homologous to PINCHand localized to focal adhesions.

PINCH1 is composed of 5 LIM domains, binds integrin-linked kinase (ILK) and locates to integrin-mediated adhesion sites. In order to investigate PINCH1 function we generated mice and embryonic stem (ES) cell-derived embryoid bodies (EBs) lacking the PINCH1 gene. Similar to mice lacking beta1 integrin or Ilk, loss of PINCH1 arrested development at the peri-implantation stage. In contrast to beta1 integrin or Ilk mutants, however, disruption of the PINCH1 gene produced implantation chambers with visible cell clumps even at embryonic day 9.5. In order to define the phenotype leading to the peri-implantation lethality we made PINCH1-null EBs and found similar but also additional defects not observed in beta1 integrin or Ilk mutant EBs. The similarities included abnormal epiblast polarity, impaired cavitation and detachment of endoderm and epiblast from basement membranes. Additional defects, which were not observed in beta1 integrin- or ILK-deficient mice or EBs, included abnormal cell-cell adhesion of endoderm and epiblast as well as the presence of apoptotic cells in the endodermal cell layer. Although ILK and PINCH1 were shown to be involved in the phosphorylation of serine-473 of PKB/Akt, immunostaining with specific antibodies revealed no apparent alteration of PKB/Akt phosphorylation in PINCH1-deficient EBs. Altogether these data demonstrate an important role of PINCH1 for integrin function, actin organization, cell-cell adhesion and endodermal cell survival during the implanting of mouse embryos.

PINCH1 regulates cell-matrix and cell-cell adhesions, cell polarity and cell survival during the peri-implantation stage.

PINCH2 belongs, together with PINCH1, to a new family of focal adhesion proteins, the members of which are composed of five LIM domains. PINCH1 and PINCH2 interact, through their first LIM domain, with the integrin-linked kinase and thereby link integrins with several signal transduction pathways. Despite their high similarity, it has been shown that PINCH1 and PINCH2 could exert distinct functions during cell spreading and cell survival. To investigate the function of PINCH2 in vivo, we deleted PINCH2 in mouse using the loxP/Cre system. In contrast to the PINCH1-deficient mice, which die at the peri-implantation stage, PINCH2-null mice are viable, fertile and show no overt phenotype. Histological analysis of tissues that express high levels of PINCH2 such as bladder and kidney revealed no apparent abnormalities, but showed a significant upregulation of PINCH1, suggesting that the two PINCH proteins may have, at least in part, overlapping function in vivo. To further test this possibility, we established PINCH1-null mouse embryonic fibroblasts, which express neither PINCH1 nor PINCH2. We found that in fibroblasts with a PINCH1/2-null background, PINCH2 is able to rescue the spreading and adhesion defects of mutant fibroblasts to the same extent as PINCH1. Furthermore, we show that the LIM1 domain only of either PINCH1 or PINCH2 can prevent ILK degradation despite their failure to localize to focal adhesions. Altogether these results suggest that PINCH1 and PINCH2 share overlapping functions and operate dependently and independently of their subcellular localization.

Consequences of loss of PINCH2 expression in mice.

An essential component of microtubules, alpha-tubulin is also a multigene family in many species. An orthology-based nomenclature for this gene family has previously been difficult to assign due to incomplete genome builds and the high degree of sequence similarity between members of this family. Using the current genome builds, sequence analysis of human, mouse, and rat alpha-tubulin genes has enabled an updated nomenclature to be generated. This revised nomenclature provides a unified language for the discussion of these genes in mammalian species; it has been approved by the gene nomenclature committees of the three species and is supported by researchers in the field.

A revised nomenclature for the human and rodent alpha-tubulin gene family.

The gene encoding cortactin, CTTN (locus 11q13), an actin-binding substrate of Src kinases, is frequently amplified in breast and head and neck squamous cell carcinomas (HNSCC) and cortactin overexpression is thought to contribute in a significant way to the invasive phenotype of these tumors. Elevated Epidermal Growth Factor receptor (EGFR) expression is also commonly observed in HNSCC and has been associated with poor prognosis and resistance to cytotoxic agents, including ionizing radiation. It has been suggested that cortactin overexpression may increase EGFR levels in these tumors by affecting receptor downregulation, however we recently found by multivariate analysis, that cortactin expression status remained an independent prognostic factor for local recurrence, disease-free survival, and overall survival.

Overexpression of cortactin in head and neck squamous cell carcinomas can be uncoupled from augmented EGF receptor expression.

Integrin-linked kinase (ILK) and cytoplasmic adaptors of the PINCH and parvin families form a ternary complex, termed IPP, that localizes to integrin adhesions. We show here that deletion of the genes encoding ILK or PINCH1 similarly blocks maturation of focal adhesions to tensin-rich and phosphotyrosine-poor fibrillar adhesions (FBs) by downregulating expression or recruitment of tensin and destabilizing alpha5beta1-integrin-cytoskeleton linkages. As IPP components are interdependent for integrin targeting and protein stability, functional dissection of the complex was achieved by fusing ILK, PINCH, parvin or their individual motifs to the cytoplasmic tail of beta3 integrin, normally excluded from FBs. Using this novel gain-of-function approach, we demonstrated that expression of the C-terminal kinase domain of ILK can restore tensin recruitment and prompt focal-adhesion maturation in IPP-null cells. Debilitating mutations in the paxillin- or ATP-binding sites of ILK, together with alpha-parvin silencing, revealed a determinant role for ILK-parvin association, but not for direct paxillin binding, in this function. We propose a model in which the C-terminal domain of ILK promotes integrin sorting by reinforcing alpha5beta1-integrin-actin linkage and controls force transmission by targeting tensin to maturing adhesions.

Molecular dissection of the ILK-PINCH-parvin triad reveals a fundamental role for the ILK kinase domain in the late stages of focal-adhesion maturation.

&#x80BF;&#x7624;&#x7EC6;&#x80DE;&#x6297;&#x7535;&#x79BB;&#x8F90;&#x5C04;&#x548C;&#x5316;&#x7597;&#x662F;&#x6CBB;&#x7597;&#x764C;&#x75C7;&#x7684;&#x4E3B;&#x8981;&#x969C;&#x788D;&#x3002;&#x4E3A;&#x6B64;&#x4F5C;&#x51FA;&#x8D21;&#x732E;&#x7684;&#x4E00;&#x4E2A;&#x56E0;&#x7D20;&#x662F;&#x5BF9; ECM &#x6574;&#x5408;&#x7D20;&#x4ECB;&#x5BFC;&#x7684;&#x9644;&#x7740;&#x529B;&#x3002;&#x7279;&#x522B;&#x6709;&#x8DA3;&#x7684;&#x65B0;&#x7EC4;&#x6C28;&#x9178;-&#x5BCC;&#x542B;&#x534A;&#x80F1;&#x6C28;&#x9178; (PINCH1) 1 &#x9002;&#x914D;&#x5668;&#x86CB;&#x767D;&#x62DB;&#x8058;&#x5230;&#x6574;&#x5408;&#x7D20;&#x9ECF;&#x9644;&#x7AD9;&#x70B9;&#x548C;&#x4FC3;&#x8FDB;&#x7EC6;&#x80DE;&#x751F;&#x5B58;&#xFF0C;&#x4F46;&#x8FD9;&#x79CD;&#x6548;&#x679C;&#x7684;&#x57FA;&#x672C;&#x7684;&#x673A;&#x5236;&#x8FD8;&#x4E0D;&#x592A;&#x6E05;&#x695A;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x5DF2;&#x7ECF;&#x8868;&#x660E; PINCH1 &#x8868;&#x793A;&#x5728;&#x4EBA;&#x7C7B;&#x80BF;&#x7624;&#x4E0E;&#x6B63;&#x5E38;&#x7EC4;&#x7EC7;&#x7684;&#x4E0D;&#x540C;&#x8D77;&#x6E90;&#x7684;&#x6C34;&#x5E73;&#x5347;&#x9AD8;&#x3002;&#x6B64;&#x5916;&#xFF0C;PINCH1&#xFF0C;&#x5EF6;&#x7EED; Akt1 &#x78F7;&#x9178;&#x5316;&#x548C;&#x6D3B;&#x52A8;&#x4FC3;&#x8FDB;&#x4F53;&#x5916;&#x548C;&#x4F53;&#x5185;&#x7684;&#x7535;&#x79BB;&#x8F90;&#x5C04;&#x5904;&#x7406;&#x540E;&#x7684;&#x7EC6;&#x80DE;&#x5B58;&#x6D3B;&#x3002;&#x673A;&#x68B0;&#x5316;&#xFF0C;PINCH1 &#x88AB;&#x53D1;&#x73B0;&#x76F4;&#x63A5;&#x7ED1;&#x5B9A;&#x5230;&#x86CB;&#x767D;&#x78F7;&#x9178;&#x9176; 1alpha (PP1alpha)-Akt1 &#x8C03;&#x8282;&#x86CB;&#x767D;&#x548C;&#x6291;&#x5236; PP1alpha &#x6D3B;&#x52A8;&#xFF0C;&#x5BFC;&#x81F4;&#x5728;&#x589E;&#x52A0; Akt1 &#x78F7;&#x9178;&#x5316;&#x548C;&#x589E;&#x5F3A;&#x6297;&#x6027;&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x6211;&#x4EEC;&#x7684;&#x6570;&#x636E;&#x8868;&#x660E;&#x9488;&#x5BF9;&#x4FE1;&#x53F7;&#x7684;&#x5206;&#x5B50;&#xFF0C;&#x4F8B;&#x5982; PINCH1 &#x4E0B;&#x6E38;&#x8BE5;&#x51FD;&#x6570;&#x7684;&#x7126;&#x7C98;&#x8FDE; &#xFF08;&#x8C03;&#x89E3; ECM &#x80BF;&#x7624;&#x7EC6;&#x80DE;&#x7C98;&#x9644;&#x6027;&#x7684;&#x914D;&#x5408;&#xFF09; &#x53EF;&#x80FD;&#x514B;&#x670D;&#x7535;&#x53F0;&#x548C;&#x5316;&#x7597;&#x8010;&#x836F;&#xFF0C;&#x4E3A;&#x764C;&#x75C7;&#x63D0;&#x4F9B;&#x65B0;&#x7684;&#x6CBB;&#x7597;&#x65B9;&#x6CD5;&#x3002;

PINCH1 &#x89C4;&#x8303; Akt1 &#x6FC0;&#x6D3B;&#xFF0C;&#x5E76;&#x901A;&#x8FC7;&#x6291;&#x5236; PP1alpha &#x589E;&#x5F3A;&#x6297;&#x6027;&#x3002;

Tumor cell resistance to ionizing radiation and chemotherapy is a major obstacle in cancer therapy. One factor contributing to this is integrin-mediated adhesion to ECM. The adapter protein particularly interesting new cysteine-histidine-rich 1 (PINCH1) is recruited to integrin adhesion sites and promotes cell survival, but the mechanisms underlying this effect are not well understood. Here we have shown that PINCH1 is expressed at elevated levels in human tumors of diverse origins relative to normal tissue. Furthermore, PINCH1 promoted cell survival upon treatment with ionizing radiation in vitro and in vivo by perpetuating Akt1 phosphorylation and activity. Mechanistically, PINCH1 was found to directly bind to protein phosphatase 1alpha (PP1alpha) - an Akt1-regulating protein - and inhibit PP1alpha activity, resulting in increased Akt1 phosphorylation and enhanced radioresistance. Thus, our data suggest that targeting signaling molecules such as PINCH1 that function downstream of focal adhesions (the complexes that mediate tumor cell adhesion to ECM) may overcome radio- and chemoresistance, providing new therapeutic approaches for cancer.

PINCH1 regulates Akt1 activation and enhances radioresistance by inhibiting PP1alpha.

R&eacute;sistance de la cellule tumorale &agrave; des rayonnements ionisants et de la chimioth&eacute;rapie est un obstacle majeur dans le traitement du cancer. Un facteur contribuant &agrave; cette est induite par l&#39;int&eacute;grine adh&eacute;rence &agrave; la MEC. Le prot&eacute;ines d&#39;adaptateur particuli&egrave;rement int&eacute;ressant nouveau histidine-riche en cyst&eacute;ine 1 (PINCH1) est recrut&eacute; aux sites d&#39;adh&eacute;sion int&eacute;grine et favorise la survie des cellules, mais les m&eacute;canismes sous-jacents &agrave; cet effet ne sont pas bien comprises. Ici, nous avons montr&eacute; que PINCH1 est exprim&eacute;e &agrave; des niveaux &eacute;lev&eacute;s dans les tumeurs humaines d&#39;origines diverses par rapport au tissu normal. En outre, PINCH1 favoris&eacute; la survie des cellules trait&eacute;s par rayonnements ionisants in vitro et in vivo en perp&eacute;tuant l&#39;activit&eacute; et la phosphorylation Akt1. M&eacute;caniquement, le PINCH1 s&#39;est av&eacute;r&eacute; pour directement lier &agrave; la prot&eacute;ine phosphatase 1alpha (PP1alpha) - une prot&eacute;ine Akt1-r&eacute;glementation - et inhiber l&#39;activit&eacute; de PP1alpha, r&eacute;sultant en accrue Akt1 phosphorylation et radior&eacute;sistance. Ainsi, nos r&eacute;sultats sugg&egrave;rent que ciblant les mol&eacute;cules de signalisation comme PINCH1 qui fonctionnent en aval des adh&eacute;rences focales (les complexes qui m&eacute;dient adh&eacute;sion cellulaire tumorale &agrave; ECM) peut surmonter les radio - et chimior&eacute;sistance, fournissant de nouvelles approches th&eacute;rapeutiques pour le cancer.

PINCH1 r&eacute;gule l&#39;activation Akt1 et am&eacute;liore la radior&eacute;sistance en inhibant la PP1alpha.

Tumor Zelle Widerstand gegen ionisierende Strahlung und Chemotherapie ist ein Haupthindernis in der Krebstherapie. Ein Faktor Beitrag dazu ist Integrin-vermittelte Adh&auml;sion zu ECM. Das Adapter-Protein besonders interessante neue Cystein-Histidin-reiche 1 (PINCH1) wird zu Integrin Adh&auml;sion Websites eingestellt und f&ouml;rdert Zelle &uuml;berleben, aber die Mechanismen dieser Effekt nicht gut verstanden. Hier haben wir gezeigt, dass PINCH1 auf den erh&ouml;hten Ebenen menschlichen Tumoren des unterschiedlichster Herkunft im Vergleich zu normalem Gewebe ausgedr&uuml;ckt wird. Dar&uuml;ber hinaus f&ouml;rderte PINCH1 Zelle &Uuml;berleben nach Behandlung mit ionisierenden Strahlen in vitro und in vivo verewigen Akt1 Phosphorylierung und Aktivit&auml;t. Mechanistisch, PINCH1 wurde festgestellt, dass direkt an Protein Phosphatase 1alpha binden (PP1alpha) - ein Akt1-Regulierung-Protein - und PP1alpha Aktivit&auml;t zu hemmen, was in erh&ouml;hten Akt1 Phosphorylierung und verbesserte Radioresistance. Unsere Daten zufolge so gezielt signalisierende Molek&uuml;le wie PINCH1 diese Funktion stromabw&auml;rts von fokalen Adh&auml;sionen (die komplexe, die Tumor-Zell-Adh&auml;sion zu ECM vermitteln) Radio- und Chemoresistance, die neue Therapieans&auml;tze f&uuml;r Krebs &uuml;berwinden kann.

PINCH1 Akt1 Aktivierung reguliert und erh&ouml;ht die Radioresistance durch die Hemmung der PP1alpha.

Resistenza delle cellule tumorali a radiazioni ionizzanti e la chemioterapia &egrave; un ostacolo importante nella terapia del cancro. Un fattore che contribuisce a questo &egrave; adesione integrina-mediata di ECM. La proteina adattatore particolarmente interessanti nuovi ricchi di cisteina-istidina 1 (PINCH1) viene reclutata per siti di adesione integrina e promuove la sopravvivenza delle cellule, ma non sono ben compresi i meccanismi alla base di questo effetto. Qui abbiamo dimostrato che PINCH1 &egrave; espressa a livelli elevati nei tumori umani di diverse origine rispetto al tessuto normale. Inoltre, PINCH1 promosso la sopravvivenza delle cellule dopo trattamento con radiazioni ionizzanti in vitro e in vivo di perpetuare le attivit&agrave; e la fosforilazione di Akt1. Meccanicistico, PINCH1 &egrave; stato trovato da associare direttamente alla proteina fosfatasi 1alpha (PP1alpha) - una proteina Akt1-regolamentare - e inibire l&#39;attivit&agrave; di PP1alpha, con conseguente aumentata fosforilazione di Akt1 ed enhanced radioresistenza. Cos&igrave;, i nostri dati suggeriscono che molecole di segnalazione come PINCH1 di targeting che funzionano a valle delle adesioni focali (i complessi che mediano l&#39;adesione delle cellule tumorali di ECM) pu&ograve; superare radio - e chemoresistance, fornendo nuovi approcci terapeutici per il cancro.

PINCH1 regola Akt1 attivazione e migliora la radioresistenza inibendo la PP1alpha.

&#x96FB;&#x96E2;&#x653E;&#x5C04;&#x7DDA;&#x3068;&#x5316;&#x5B66;&#x7642;&#x6CD5;&#x306B;&#x816B;&#x760D;&#x306E;&#x30BB;&#x30EB;&#x62B5;&#x6297;&#x764C;&#x6CBB;&#x7642;&#x306B;&#x304A;&#x3051;&#x308B;&#x4E3B;&#x306A;&#x969C;&#x5BB3;&#x3067;&#x3059;&#x3002;&#x3053;&#x308C;&#x306B;&#x8CA2;&#x732E;&#x3057;&#x3066;&#x4E00;&#x3064;&#x306E;&#x8981;&#x56E0;&#x306F;&#x3001;ECM &#x3078;&#x306E;&#x30A4;&#x30F3;&#x30C6;&#x30B0;&#x30EA;&#x30F3;&#x3092;&#x4ECB;&#x3057;&#x305F;&#x63A5;&#x7740;&#x3067;&#x3059;&#x3002;&#x7279;&#x306B;&#x8208;&#x5473;&#x6DF1;&#x3044;&#x65B0;&#x3057;&#x3044;&#x30B7;&#x30B9;&#x30C6;&#x30A4;&#x30F3;-&#x30D2;&#x30B9;&#x30C1;&#x30B8;&#x30F3;&#x8C4A;&#x5BCC;&#x306A; 1 (PINCH1) &#x30A2;&#x30C0;&#x30D7;&#x30BF;&#x30FC;&#x86CB;&#x767D;&#x8CEA;&#x30A4;&#x30F3;&#x30C6;&#x30B0;&#x30EA;&#x30F3;&#x63A5;&#x7740;&#x30B5;&#x30A4;&#x30C8;&#x3092;&#x52DF;&#x96C6;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3001;&#x30BB;&#x30EB;&#x5B58;&#x7D9A;&#x3092;&#x4FC3;&#x9032;&#x3059;&#x308B;&#x304C;&#x3001;&#x3053;&#x306E;&#x52B9;&#x679C;&#x306E;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x306F;&#x3088;&#x304F;&#x7406;&#x89E3;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x305B;&#x3093;&#x3002;&#x3053;&#x3053;&#x3067; PINCH1 &#x6B63;&#x5E38;&#x7D44;&#x7E54;&#x306B;&#x5BFE;&#x3059;&#x308B;&#x591A;&#x69D8;&#x306A;&#x8D77;&#x6E90;&#x306E;&#x3072;&#x3068;&#x816B;&#x760D;&#x306B;&#x304A;&#x3051;&#x308B;&#x7BA1;&#x7406;&#x8005;&#x7279;&#x6A29;&#x30EC;&#x30D9;&#x30EB;&#x3067;&#x8868;&#x73FE;&#x3055;&#x308C;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;PINCH1 &#x306B;&#x3088;&#x308B; in vitro &#x304A;&#x3088;&#x3073; in vivo &#x3067;&#x306E;&#x96FB;&#x96E2;&#x653E;&#x5C04;&#x7DDA;&#x6CBB;&#x7642;&#x6642;&#x306E;&#x7D30;&#x80DE;&#x306E;&#x751F;&#x5B58; Akt1 &#x306E;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x3068;&#x6D3B;&#x52D5;&#x3092;&#x6C38;&#x7D9A;&#x3055;&#x305B;&#x308B;&#x306B;&#x6607;&#x683C;&#x3002;&#x6A5F;&#x68B0;&#x8AD6;&#x7684;&#x306B;&#x3001;PINCH1 &#x306F;&#x76F4;&#x63A5;&#x86CB;&#x767D;&#x8CEA;&#x30DB;&#x30B9;&#x30D5;&#x30A1;&#x30BF;&#x30FC;&#x30BC; 1 &alpha; &#x306B;&#x30D0;&#x30A4;&#x30F3;&#x30C9;&#x304C;&#x898B;&#x3064;&#x304B;&#x308A;&#x307E;&#x305B;&#x3093;&#x3067;&#x3057;&#x305F; (PP1alpha)-&#x3001;Akt1 &#x8ABF;&#x7BC0;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA; - &#x3068; PP1alpha &#x6D3B;&#x6027;&#x3092;&#x963B;&#x5BB3;&#x3001;&#x7D50;&#x679C;&#x306B; Akt1 &#x306E;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x3092;&#x5897;&#x52A0;&#x3057;&#x3001;&#x653E;&#x5C04;&#x7DDA;&#x8010;&#x6027;&#x3092;&#x5F37;&#x5316;&#x3057;&#x307E;&#x3059;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;&#x6211;&#x3005; &#x306E;&#x30C7;&#x30FC;&#x30BF;&#x306F; PINCH1 &#x306A;&#x3069;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x5206;&#x5B50;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x95A2;&#x6570;&#x4E0B;&#x6D41;&#x30A4;&#x30F3;&#x30C6;&#x30B0;&#x30EA;&#x30F3;&#x30EA;&#x30F3;&#x30AF;&#x30C9;&#x30AD;&#x30CA;&#x30FC;&#x30BC; &#xFF08;ECM &#x3078;&#x306E;&#x816B;&#x760D;&#x7D30;&#x80DE;&#x63A5;&#x7740;&#x3092;&#x4EF2;&#x4ECB;&#x3059;&#x308B;&#x932F;&#x4F53;) &#x306E;&#x30E9;&#x30B8;&#x30AA;&#x304A;&#x3088;&#x3073; chemoresistance &#x3092;&#x7570;&#x306A;&#x308A;&#x3001;&#x304C;&#x3093;&#x306E;&#x65B0;&#x3057;&#x3044;&#x6CBB;&#x7642;&#x306E;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x3092;&#x63D0;&#x4F9B;&#x3059;&#x308B;&#x514B;&#x670D;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x304A;&#x52E7;&#x3081;&#x3057;&#x307E;&#x3059;&#x3002;

PINCH1 Akt1 &#x306E;&#x6D3B;&#x6027;&#x5316;&#x3092;&#x8ABF;&#x7BC0;&#x3057;&#x3066; PP1alpha &#x3092;&#x6291;&#x5236;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x3001;&#x653E;&#x5C04;&#x7DDA;&#x8010;&#x6027;&#x3092;&#x9AD8;&#x3081;&#x307E;&#x3059;&#x3002;

&#xC885;&#xC591; &#xC138;&#xD3EC; &#xC800;&#xD56D; &#xC804;&#xB9AC; &#xBC29;&#xC0AC;&#xC120; &#xBC0F; &#xD654;&#xD559; &#xC694;&#xBC95;&#xC744; &#xC554; &#xCE58;&#xB8CC;&#xC5D0; &#xC911;&#xC694; &#xD55C; &#xC7A5;&#xC560; &#xC774;&#xB2E4;. &#xC774;&#xC5D0; &#xAE30;&#xC5EC; &#xD558;&#xB294; &#xC694;&#xC18C; &#xC911; &#xD558;&#xB098;&#xB294; ECM&#xC5D0; &#xC811;&#xCC29; &#xB825;&#xC744; integrin &#xC911;&#xC7AC;&#xC785;&#xB2C8;&#xB2E4;. &#xD2B9;&#xD788; &#xC0C8; &#xC2DC;&#xC2A4;&#xD14C;&#xC778;-&#xD788;&#xC2A4;&#xD2F0;&#xB518;-&#xB9AC;&#xCE58; 1 (PINCH1) &#xC7AC;&#xBBF8; &#xC788;&#xB294; &#xC5B4;&#xB311;&#xD130; &#xB2E8;&#xBC31;&#xC9C8; integrin &#xC811;&#xCC29; &#xC0AC;&#xC774;&#xD2B8;&#xC5D0; &#xBCF4;&#xCDA9; &#xBC0F; &#xC138;&#xD3EC; &#xC0DD;&#xC874;&#xC744; &#xCD09;&#xC9C4; &#xD558;&#xC9C0;&#xB9CC;&#xC774; &#xD6A8;&#xACFC; &#xAE30;&#xBCF8; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC744; &#xC798; &#xC774;&#xD574; &#xB418;&#xC9C0; &#xC54A;&#xB294;&#xB2E4;. &#xC5EC;&#xAE30; &#xC6B0;&#xB9AC; PINCH1 &#xC815;&#xC0C1; &#xC870;&#xC9C1;&#xC5D0; &#xC0C1;&#xB300;&#xC801;&#xC73C;&#xB85C; &#xB2E4;&#xC591; &#xD55C; &#xAE30;&#xC6D0;&#xC758; &#xC778;&#xAC04; &#xC885;&#xC591;&#xC758; &#xB192;&#xC740; &#xC218;&#xC900;&#xC5D0;&#xC11C; &#xD45C;&#xC2DC; &#xB429;&#xB2C8;&#xB2E4; &#xAC83;&#xC73C;&#xB85C; &#xB098;&#xD0C0;&#xB0AC;&#xC2B5;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, PINCH1 Akt1 &#xC778; &#xC0B0;&#xD654; &#xBC0F; &#xD65C;&#xB3D9;&#xC744; &#xC601; &#xC18D;&#xC5D0; &#xC758;&#xD574; &#xC0DD;&#xCCB4; &#xC678; &#xBC0F; &#xC0DD;&#xCCB4; &#xC870;&#xAC74; &#xC804;&#xB9AC; &#xBC29;&#xC0AC;&#xC120; &#xCE58;&#xB8CC; &#xC2DC; &#xC138;&#xD3EC; &#xC0DD;&#xC874;&#xC744; &#xC2B9;&#xC9C4;. Mechanistically, PINCH1 &#xB2E8;&#xBC31;&#xC9C8; &#xC778;&#xC0B0; &#xAC00;&#xC218;&#xBD84;&#xD574; &#xD6A8;&#xC18C; 1alpha&#xC5D0; &#xC9C1;&#xC811; &#xBC14;&#xC778;&#xB529;&#xD560; &#xCC3E;&#xC558;&#xC2B5;&#xB2C8;&#xB2E4; (PP1alpha)-Akt1 &#xC870;&#xC808; &#xB2E8;&#xBC31;&#xC9C8;-PP1alpha &#xD65C;&#xB3D9;&#xC744; &#xC5B5;&#xC81C; &#xD558; &#xACE0;, &#xACB0;&#xACFC; Akt1 &#xC778; &#xC0B0;&#xD654;&#xB97C; &#xC99D;&#xAC00; &#xBC0F; &#xD5A5;&#xC0C1; &#xB41C; radioresistance. &#xB530;&#xB77C;&#xC11C;, &#xC6B0;&#xB9AC;&#xC758; &#xB370;&#xC774;&#xD130;&#xB294; PINCH1 &#xB4F1; &#xC2E0;&#xD638; &#xBD84;&#xC790;&#xB97C; &#xB300;&#xC0C1;&#xC73C;&#xB85C; &#xD568;&#xC218; &#xD558;&#xB958; &#xCD08;&#xC810; &#xC720;&#xCC29; (ECM&#xC5D0; &#xC885;&#xC591; &#xC138;&#xD3EC; &#xC811;&#xCC29; &#xC911;&#xC7AC; &#xB2E8;&#xC9C0;)&#xC758; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xADF9;&#xBCF5; &#xB77C;&#xB514;&#xC624; &#xBC0F; chemoresistance, &#xC554;&#xC5D0; &#xB300; &#xD55C; &#xC0C8;&#xB85C;&#xC6B4; &#xCE58;&#xB8CC; &#xC811;&#xADFC;&#xC744; &#xC81C;&#xACF5; &#xD558; &#xACE0; &#xAC83;&#xC774; &#xC88B;&#xC2B5;&#xB2C8;&#xB2E4;.

PINCH1 Akt1 &#xD65C;&#xC131;&#xD654;&#xB97C; &#xC870;&#xC808; &#xD558; &#xACE0; pp1alpha&#xC744; &#xC5B5;&#xC81C; &#xD568;&#xC73C;&#xB85C;&#xC368; radioresistance&#xC744; &#xD5A5;&#xC0C1; &#xC2DC;&#xD0B5;&#xB2C8;&#xB2E4;.

Tumor celular resistencia a la radiaci&oacute;n ionizante y la quimioterapia es un obst&aacute;culo importante en el tratamiento del c&aacute;ncer. Un factor que contribuye a esto es adherencia mediada por integrinas a ECM. La prote&iacute;na de adaptador particularmente interesante a nuevo ciste&iacute;na-histidina-rico 1 (PINCH1) es reclutada para sitios de adherencia de integrina y promueve la supervivencia de la c&eacute;lula, pero los mecanismos subyacentes a este efecto no son bien entendidos. Aqu&iacute; hemos demostrado que la PINCH1 se expresa en niveles elevados en los tumores humanos de diversos or&iacute;genes en relaci&oacute;n con el tejido normal. Adem&aacute;s, PINCH1 hab&iacute;a promovido supervivencia celular sobre el tratamiento con radiaci&oacute;n ionizante in vitro e in vivo por perpetuar la actividad y Akt1 fosforilaci&oacute;n. Mec&aacute;nico, PINCH1 fue encontrado enlazar directamente a la prote&iacute;na fosfatasa 1alpha (PP1alpha) - una prote&iacute;na reguladora Akt1 - e inhiben la actividad de PP1alpha, resultando en mayor fosforilaci&oacute;n de Akt1 y mejorado radioresistencia. As&iacute;, nuestros datos sugieren que a mol&eacute;culas de se&ntilde;alizaci&oacute;n como PINCH1 esa funci&oacute;n aguas abajo de adherencias focales (los complejos que median la adhesi&oacute;n de c&eacute;lulas de tumor a ECM) puede superar radio - y quimiorresistencia, proporcionando nuevos enfoques terap&eacute;uticos para el c&aacute;ncer.

PINCH1 regula la activaci&oacute;n de Akt1 y mejora la radioresistencia inhibiendo PP1alpha.

Actin filaments are instrumental in driving processes such as migration, cytokinesis and endocytosis and provide cells with mechanical support. During angiogenesis, actin-rich filopodia protrusions have been proposed to drive endothelial tip cell functions by translating guidance cues into directional migration and mediating new contacts during anastomosis. To investigate the structural organisation, dynamics and functional importance of F-actin in endothelial cells (ECs) during angiogenesis in vivo, we generated a transgenic zebrafish line expressing Lifeact-EGFP in ECs. Live imaging identifies dynamic and transient F-actin-based structures, such as filopodia, contractile ring and cell cortex, and more persistent F-actin-based structures, such as cell junctions. For functional analysis, we used low concentrations of Latrunculin B that preferentially inhibited F-actin polymerisation in filopodia. In the absence of filopodia, ECs continued to migrate, albeit at reduced velocity. Detailed morphological analysis reveals that ECs generate lamellipodia that are sufficient to drive EC migration when filopodia formation is inhibited. Vessel guidance continues unperturbed during intersegmental vessel development in the absence of filopodia. Additionally, hypersprouting induced by loss of Dll4 and attraction of aberrant vessels towards ectopic sources of Vegfa165 can occur in the absence of endothelial filopodia protrusion. These results reveal that the induction of tip cells and the integration of endothelial guidance cues do not require filopodia. Anastomosis, however, shows regional variations in filopodia requirement, suggesting that ECs might rely on different protrusive structures depending on the nature of the environment or of angiogenic cues.

Filopodia are dispensable for endothelial tip cell guidance.

The spatiotemporal and longitudinal monitoring of cellular processes occurring in tumors is critical for oncological research. We focused on glioblastoma multiforme (GBM), an untreatable highly vascularized brain tumor whose progression is thought to critically depend on the oxygen and metabolites supplied by blood vessels. We optimized protocols for orthotopic GBM grafting in mice that were able to recapitulate the biophysical constraints normally governing tumor progression and were suitable for intravital multiphoton microscopy. We repeatedly imaged tumor cells and blood vessels during GBM development. We established methods for quantitative correlative analyses of dynamic imaging data over wide fields in order to cover the entire tumor. We searched whether correlations existed between blood vessel density, tumor cell density and proliferation in control tumors. Extensive vascular remodeling and the formation of new vessels accompanied U87 tumor cell growth, but no strong correlation was found between local cell density and the extent of local blood vessel density irrespective of the tumor area or time points. The technique moreover proves useful for comparative analysis of mice subjected either to Bevacizumab anti-angiogenic treatment that targets VEGF or to AMD3100, an antagonist of CXCR4 receptor. Bevacizumab treatment massively reduced tumoral vessel densities but only transiently reduced U87 tumor growth rate. Again, there was no correlation between local blood vessel density and local cell density. Moreover, Bev applied only prior to tumor implantation inhibited tumor growth to the same extent as post-grafting treatment. AMD3100 achieved a potent inhibition of tumor growth without significant reduction in blood vessel density. These results indicate that in the brain, in this model, tumor growth can be sustained without an increase in blood vessel density and suggest that GBM growth is rather governed by stromal properties.

Dynamic quantitative intravital imaging of glioblastoma progression reveals a lack of correlation between tumor growth and blood vessel density.

Endothelial cells show surprising cell rearrangement behaviour during angiogenic sprouting; however, the underlying mechanisms and functional importance remain unclear. By combining computational modelling with experimentation, we identify that Notch/VEGFR-regulated differential dynamics of VE-cadherin junctions drive functional endothelial cell rearrangements during sprouting. We propose that continual flux in Notch signalling levels in individual cells results in differential VE-cadherin turnover and junctional-cortex protrusions, which powers differential cell movement. In cultured endothelial cells, Notch signalling quantitatively reduced junctional VE-cadherin mobility. In simulations, only differential adhesion dynamics generated long-range position changes, required for tip cell competition and stalk cell intercalation. Simulation and quantitative image analysis on VE-cadherin junctional patterning in vivo identified that differential VE-cadherin mobility is lost under pathological high VEGF conditions, in retinopathy and tumour vessels. Our results provide a mechanistic concept for how cells rearrange during normal sprouting and how rearrangement switches to generate abnormal vessels in pathologies.

Fabio Stanchi

Developmental Biology Institute of Marseille

Aix Marseille University

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Fabio Stanchi

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Developmental Biology Institute of Marseille

Aix Marseille University

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Developmental Biology Institute of Marseille