Literatura académica sobre el tema "ITGB3"

Ovarian folliculogenesis and oogenesis has a significant impact on embryo growth and development in preimplantation stages. Although both processes are widely understood in several species of mammals, including pigs, the factors influencing the proper maturation capability of oocytes, as well as the developmental competence of the surrounding somatic granulosa cells (GCs) and cumulus cells (CCs), are still not entirely known. This study aimed to investigate the expression of growth differentiation factor 9 (GDF9) and integrins (ITGB1, ITGB2, ITGB3 and ITGB4) in porcine oocytes isolated from follicles of various size and donors characterized by different puberty status. The relative abundance of GDF9, ITGB1, ITGB2, ITGB3, and ITGB4 mRNAs in porcine oocytes isolated from medium follicles of cycling sows (MFCS), small follicles of juvenile gilts (SFJG), and small follicles of cycling sows (SFCS) was assessed by an RT-qPCR assay. We found an increased expression of GDF9 in oocytes isolated from the small follicles of juvenile gilts as compared to the other two groups (P<0.001). A significant down-regulation of ITGB1 and ITGB2 oocyte mRNAs collected from medium follicles of cycling sows was observed (P<0.05 and P<0.001, respectively). The ITGB3 mRNA expression was significantly decreased in oocytes isolated from small follicles of juvenile gilts (P<0.001), whereas a lower expression of ITGB4 in oocytes from both medium follicles (cycling sows) and small follicles (juvenile gilts) was observed. In conclusion, GDF9 may be recognized as the main factor regulating follicle growth at early stages of folliculogenesis. The expression of ITGBs is significantly regulated by the puberty status of donor pigs, and different follicular sizes may play a subordinate role in integrin expression during in vivo follicle development in pigs.

The process of implantation is mediated by a complex network of signaling and adhesive factors. In the pig, latent and active transforming growth factor beta (TGFB), TGFB receptors (TGFBR), and integrins (ITGs) are present during the peri-implantation period. TGFB signals via TGFBR and activates downstream effector SMAD proteins 2 and 3 (p-SMAD2/3). Latency-associated peptide (LAP), part of the latent TGFB complex, is known to bind to ITG heterodimers and activate TGFB. We hypothesize that active TGFBs and TGFBRs along with LAP and ITGs functionally interact at the conceptus–maternal interface to mediate events essential for conceptus development and attachment in pigs. Uteri and conceptuses from days 10, 12, 16, 20, and 24 pregnant gilts were immunostained for TGFB, LAP, and ITG subunits (ITGAV, ITGB1, ITGB3, ITGB5, ITGB6, and ITGB8). Activation of TGFBRs was evaluated by the presence of phosphorylated downstream effector SMAD2/3. Binding of LAP to ITGs was also evaluated using porcine trophectoderm cells. Abundant active TGFB was detected at the apical surfaces of epithelia at the conceptus–maternal interface, and p-SMAD2/3 was detected at both conceptus attachment and nonattachment sites during implantation. Separate aggregates of LAP, ITGB1, ITGB5, and later ITGB3 were detected at the porcine conceptus–maternal interface, and binding of LAP to ITGs on apical surfaces was demonstrated. Results suggest that functional LAP–ITG adhesion complexes support conceptus attachment and promote TGFB activation leading to TGFB interaction with TGFBR supporting events of porcine implantation.

Integrins regulate adhesion at the foeto-maternal interface by interacting with secreted phosphoprotein 1 (SPP1) and fibronectin (FN). It is hypothesised that impaired foetal growth of ‘runt’ piglets is linked to altered integrin signalling at the foeto-maternal interface. Placental and endometrial samples associated with the lightest and closest to mean litter weight (CTMLW) (gestational day (GD18, 30, 45, 60 and 90), of both sex (GD30, 45, 60 and 90) (n = 5–8 litters/GD), Large White × Landrace conceptuses or foetuses were obtained. The mRNA expression of the integrin subunits (ITG) ITGA2, ITGAV, ITGB1, ITGB3, ITGB5, ITGB6, ITGB8, SPP1 and FN was quantified by qPCR. Temporal changes in mRNA expression were observed, with different profiles in the two tissues. Endometrial ITGB1 (P ≤ 0.05, GD45) and SPP1 (P ≤ 0.05, all GD combined and GD60) expression was decreased in samples supplying the lightest compared to the CTMLW foetuses. Placentas supplying female foetuses had decreased expression of ITGB6 (GD45, P ≤ 0.05) and FN (GD90, P ≤ 0.05) compared to those supplying male foetuses. Endometrial samples supplying females had increased ITGB3 (P ≤ 0.05, GD60) and FN (P ≤ 0.05, GD30) expression and decreased SPP1 (P ≤ 0.05, GD60) expression compared to male foetuses. Correlations between mean within-gilt mRNA expression and percentage prenatal survival, number of live foetuses or conceptuses and percentage male foetuses were observed. This study has highlighted novel and dynamic associations between foetal size, sex and integrin subunit mRNA expression at the porcine foeto-maternal interface. Further studies should be performed to improve the understanding of the mechanisms behind these novel findings.

Here, as a basic study in the construction of a non-cellular niche that supports artificial organization of three-dimensional endometrial tissue, we defined the types of integrin heterodimers that are expressed transcriptionally, translationally and functionally in endometrial stromal (ES) and endometrial epithelial (EE) cells isolated from the mouse uterus in estrus. Gene and protein expression of integrin subunits were analyzed at the transcriptional and translational level by real-time PCR and fluorescent immunoassay, respectively. Moreover, the functionality of integrin heterodimers was confirmed by attachment and antibody inhibition assays. Itga2, Itga5, Itga6, Itga9, Itgav, Itgb1, Itgb3 and Itgb5 in ES cells, and Itga2, Itga5, Itga6, Itga7, Itga9, Itgav, Itgb1, Itgb3, Itgb4, Itgb5 and Itga6 and in EE cells showed significantly higher transcriptional levels than the other integrin subunits. Furthermore, translational expression of the total integrin α and β subunit genes that showed increased transcription was determined in ES and EE cells. ES cells showed significantly increased adhesion to collagen I, fibronectin and vitronectin, and functional blocking of integrin α2, α5 or αV significantly inhibited adhesion to these molecules. Moreover, EE cells showed significantly increased adhesion to collagen I, fibronectin, laminin and vitronectin, and functional blocking of integrin α2, α5, α6 or αV significantly inhibited adhesion to these molecules. Accordingly, we confirmed that integrin α2β1, α5β1, αVβ1, αVβ3 and/or αVβ5, and integrin α2β1, α5β1, α6β1 and/or α6β4, αVβ1, αVβ3 and/or αVβ5, actively function on the surface of ES and EE cells from mouse uterus in estrus phase, respectively.

Context: The RET receptor tyrosine kinase is an important mediator of several human diseases, most notably of neuroendocrine cancers. These diseases are characterized by aberrant cell migration, a process tightly regulated by integrins. Objective: Our goals were to investigate the role of integrins in RET-mediated migration in two neoplastic cell models: the neural-derived cell line SH-SY5Y, and the papillary thyroid carcinoma cell line TPC-1. We also evaluated whether multiple integrin subunits have a role in RET-mediated cell migration. Design: We evaluated the expression and activation of integrins in response to RET activation using standard cell adhesion and migration (wound-healing) assays. We examined focal adhesion formation, using integrin-paxillin coimmunoprecipitations and immunofluorescence, as an indicator of integrin activity. Results: Our data indicate that β1 integrin (ITGB1) is expressed in both SH-SY5Y and TPC-1 cell lines and that these cells adhere strongly to matrices preferentially associated with ITGB1. We showed that RET can activate ITGB1, and that RET-induced cell adhesion and migration require ITGB1. Furthermore, we showed that β3 integrin (ITGB3) also plays a role in RET-mediated cell adhesion and migration in vitro and ITGB3 expression correlates with RET-mediated invasion in a mouse tumor xenograft model, suggesting that RET mediates the activity of multiple integrin subunits. Conclusions: Our data are the first to show that multiple integrin subunits contribute to cell adhesion and migration downstream of RET, suggesting that coordinated signaling through these pathways is important for cell interactions with the microenvironment during tumor invasion and progression.

Integrin β3 (ITGB3), which is the target gene of the miRNA let-7 that can be antagonized by long noncoding RNA (lncRNA) H19, is well known to have a critical role in endometrium receptivity. However, the regulation of ITGB3 in cell–cell or cell–extracellular matrix adhesion and invasion for the maintenance of early pregnancy remains unknown. This study aimed to explore the role of the H19/let-7/ITGB3 axis in regulating trophoblastic spheroid adhesion and in vitro invasion ability using the HTR-8/SVneo cell line and to investigate the expression levels of lncRNA H19 and ITGB3 in human products of conception. The in vitro knockdown of H19 resulted in decreased expression of ITGB3 at the mRNA and protein levels and reduced the adhesion and invasion ability. In the embryonic chorion tissue of spontaneous abortion (SA), the expressions of H19 and ITGB3 at both the mRNA and protein levels decreased. The results of quantitative RT-PCR, Western blot analysis, dual-luciferase report gene and functional miRNA let-7 rescue experiments, adhesion assay and in vitro transwell invasion assay confirmed that H19 regulated trophoblastic spheroid adhesion with endometrial stromal cells through the H19/let-7/ITGB3 axis, thereby providing an improved understanding of the molecular mechanism of SA.

Abstract Abstract 758 Primary leukemia stem cells (LSCs) reside in an in vivo microenvironment that supports the growth and survival of malignant cells. Despite the increasing understanding of the importance of niche interactions and primary cell biology in leukemia, many studies continue to focus on cell autonomous processes in artificial model systems. The majority of strategies to-date that attempt to define therapeutic targets in leukemia have relied on screening cell lines in culture; new strategies should incorporate the use of primary disease within a physiologic niche. Using a primary murine MLL-AF9 acute myeloid leukemia (AML) model highly enriched for LSCs, we performed an in vivo short hairpin RNA (shRNA) screen to identify novel genes that are essential for leukemia growth and survival. LSCs infected with pools of shRNA lentivirus were transplanted and grown in recipient mice for 2 weeks, after which bone marrow and spleen cells were isolated. Massively parallel sequencing of infected LSCs isolated before and after transplant was used to quantify the changes in shRNA representation over time. Our in vivo screens were highly sensitive, robust, and reproducible and identified a number of positive controls including genes required for MLL-AF9 transformation (Ctnnb1, Mef2c, Ccna1), genes universally required for cell survival (Ube2j2, Utp18), and genes required in other AML models (Myb, Pbx1, Hmgb3). In our primary and validation screens, multiple shRNAs targeting Integrin Beta 3 (Itgb3) were consistently depleted by more than 20-fold over two weeks in vivo. Follow up studies using RNA interference (RNAi) and Itgb3−/− mice identified Itgb3 as essential for murine leukemia cells growth and transformation in vivo, and loss of Itgb3 conferred a statistically significant survival advantage to recipient mice. Importantly, neither Itgb3 knockdown or genetic loss impaired normal hematopoietic stem and progenitor cell (HSPC) function in 16 week multilineage reconstitution assays. We further identified Itgav as the heterodimeric partner of Itgb3 in our model, and found that knockdown of Itgav inhibited leukemia cell growth in vivo. Consistent the therapeutic aims or our study, flow cytometry on primary human AML samples revealed ITGAV/ITGB3 heterodimer expression. To functionally assess the importance of gene expression in a human system, we performed another RNAi screen on M9 leukemia cells, primary human cord blood CD34+ cells transduced with MLL-ENL that are capable of growing in vitro or in a xenotransplant model in vivo. We found that ITGB3 loss inhibited M9 cell growth in vivo, but not in vitro, consistent with the importance of ITGB3 in a physiologic microenvironment. We explored the signaling pathways downstream of Itgb3 using an additional in vivo, unbiased shRNA screen and identified Syk as a critical mediator of Itgb3 activity in leukemia. Syk knockdown by RNAi inhibited leukemia cell growth in vivo; downregulation of Itgb3 expression resulted in decreased levels of Syk phosphorylation; and expression of an activated form of Syk, TEL-SYK, rescued the effects of Itgb3 knockdown on leukemia cell growth in vivo. To understand cellular processes controlled by Itgb3, we performed gene expression studies and found that, in leukemia cells, Itgb3 knockdown induced differentiation and inhibited multiple previously published LSC transcriptional programs. We confirmed these results using primary leukemia cell histology and a model system of leukemia differentiation. Finally, addition of a small molecule Syk inhibitor, R406, to primary cells co-cultured with bone marrow stroma caused a dose-dependent decrease in leukemia cell growth. Our results establish the significance of the Itgb3 signaling pathway, including Syk, as a potential therapeutic target in AML, and demonstrate the utility of in vivo RNA interference screens. Disclosures: Armstrong: Epizyme: Consultancy.

To some extent, the use of metformin may improve endometrial receptivity and pregnancy outcomes of women with polycystic ovarian syndrome (PCOS) undergoing in vitro fertilization/intracytoplasmic sperm injection. However, the mechanism is not well-known. The endometrium of metformin-treated group (metformin-treated patients with PCOS) and the control group (non-metformin-treated patients with PCOS) were analyzed for the expression of homeobox A10 (HOXA10) and integrin beta-3 (ITGB3) and differential micro RNA (miRNA) expression profiles. On this basis, miRDB and Target Scan databases were used to predict and screen out that miR-491-3p and miR-1910-3p may target HOXA10 and ITGB3. Furthermore, we verified the effects of metformin on the expression of HOXA10 and ITGB3, and regulatory effects of miR-1910-3p and miR-491-3p on HOXA10 and ITGB3 using Ishikawa cell line. Metformin induced a significant dose-dependent upregulation of HOXA10 and ITGB3. The results from the microarray analyses showed there were 40 differentially expressed miRNAs between the 2 groups. Among them, miR-1910-3p and miR-491-3p were the 2 significantly downregulated miRNAs. Bioinformatics prediction indicated that HOXA10 and ITGB3 are potential target genes for miR-1910-3p and miR-491-3p. In Ishikawa cells transfected with miR-491-3p mimics, the expression of HOXA10 and ITGB3 on both messenger RNA (mRNA) and protein level were lower than those in control group ( P < .001). Also, the expression of HOXA10 mRNA and protein was lower in Ishikawa cells transfected with miR-1910-3p mimics ( P < .001). However, no significant changes in ITGB3 levels were observed in cells transfected with miR-1910-3p mimics ( P > .05). Metformin likely improves endometrial receptivity through downregulating the expression of miR-491-3p and miR-1910-3p, thereby increasing the expression of HOXA10 and ITGB3 in the endometrium of PCOS women.

Abstract Angiogenic factors play a key role in multiple myeloma (MM) growth, relapse, and drug resistance. Here we show that malignant plasma cells (cell lines and patient-derived MM cells) express angiocrine factor EGF like-7 (EGFL7) mRNA and protein. MM cells both produced EGFL7 and expressed the functional EGFL7 receptor integrin β 3 (ITGB3), resulting in ITGB3 phosphorylation and focal adhesion kinase activation. Overexpression of ITGB3 or EGFL7 enhanced MM cell adhesion and proliferation. Intriguingly, ITGB3 overexpression upregulated the transcription factor Krüppel-like factor 2 (KLF2), which further enhanced EGFL7 transcription in MM cells, thereby establishing an EGFL7-ITGB3-KLF2-EGFL7 amplification loop that supports MM cell survival and proliferation. EGFL7 expression was found in certain plasma cells of patients with refractory MM and of patients at primary diagnosis. NOD.CB17-Prkdc&lt;scid&gt;/J mice transplanted with MM cells showed elevated human plasma EGFL7 levels. EGFL7 knockdown in patient-derived MM cells and treatment with neutralizing antibodies against EGFL7 inhibited MM cell growth in vitro and in vivo. We demonstrate that the standard-of-care MM drug bortezomib upregulates EGFL7, ITGB3, and KLF2 expression in MM cells. Inhibition of EGFL7 signaling in synergy with BTZ may provide a novel strategy for inhibiting MM cell proliferation.

The integrity of the fetal–maternal interface is critical for proper fetal nourishment during pregnancy. Integrins are important adhesion molecules present at the interface during implantation; however,in vivoevidence for integrin activation and focal adhesion formation at the maternal–conceptus interface is limited. We hypothesized that focal adhesion assembly in uterine luminal epithelium (LE) and conceptus trophectoderm (Tr) results from integrin binding of extracellular matrix (ECM) at this interface to provide increased tensile forces and signaling to coordinate utero-placental development. An ovine model of unilateral pregnancy was used to evaluate mechanotransduction events leading to focal adhesion assembly at the maternal–conceptus interface and within the uterine wall. Animals were hysterectomized on days 40, 80, or 120 of pregnancy, and uteri immunostained for integrins (ITGAV, ITGA4, ITGA5, ITGB1, ITGB3, and ITGB5), ECM proteins (SPP1, LGALS15, fibronectin (FN), and vitronectin (VTN)), cytoskeletal molecules (ACTN and TLN1), and a signal generator (PTK2). Focal adhesion assembly in myometrium and stroma was also studied to provide a frame of reference for mechanical stretch of the uterine wall. Large focal adhesions containing aggregates of ITGAV, ITGA4, ITGA5, ITGB1, ITGB5, ACTN, and PTK2 were detected in interplacentomal uterine LE and Tr of gravid but not non-gravid uterine horns and increased during pregnancy. SPP1 and LGALS15, but not FN or VTN, were present along LE and Tr interfaces in both uterine horns. These data support the idea that focal adhesion assembly at the maternal–conceptus interface reflects adaptation to increasing forces caused by the growing fetus. Cooperative binding of multiple integrins to SPP1 deposited at the maternal–conceptus interface forms an adhesive mosaic to maintain a tight connection between uterine and placental surfaces along regions of epitheliochorial placentation in sheep.

L’identification des gènes impliqués dans les thrombopénies apporte des éléments importants pour la compréhension des voies de régulation de la production et des fonctions des plaquettes voire de l’hématopoïèse. Notre laboratoire a développé une stratégie d’identification de gènes à l’origine de thrombopénies sans hypothèse a priori par séquençage d’exomes. Cette stratégie a permis de mettre en évidence des mutations touchant les gènes ETV6, ITGA2B et ITGB3.Les thrombopénies à volume plaquettaire normal sont particulièrement importantes à détecter en raison d’un risque d’évolution vers une pathologie onco-hématologique. L’origine génétique de cette catégorie de thrombopénie s’est pendant longtemps limitée aux mutations du gène runx1. Le travail que j’ai effectué au cours de ma thèse a contribué à impliquer le gène etv6 dans ce groupe de thrombopénies.Concernant le gène etv6 6 familles ont présenté des mutations pathogènes. Toutes ces mutations sont à l’origine d’une perte de l’activité répressive du gène et un nombre élevé de cellules CD34+ circulant dans le sang révélant le rôle d’ETV6 dans la prédisposition onco-hématologique. De plus la mégacaryopoïèse présente deux principales anomalies. Elles associent une augmentation du nombre de colonies progéniteurs mégacaryocytaires à une formation de proplaquettes réduites. Concernant les gènes itga2b et itgb3 3 familles ont été étudiées.Des défauts quantitatifs ou qualitatifs du récepteur αIIbβ3 conduisant à sa perte de fonction se retrouvent dans la thrombasthénie de Glanzmann (GT) caractérisée par une thrombopathie mais un nombre de plaquettes et une morphologie normale
The identification of the genes involved in thrombocytopenia provides important elements for understanding the pathways of regulation of the production and functions of platelets or even hematopoiesis. Our laboratory has developed a strategy for identifying genes causing thrombocytopenia without a priori hypothesis by sequencing exomes. This strategy has been applied to families with autosomal dominant thrombocytopenia and has demonstrated mutations in the genes etv6, ​​itga2b and itgb3. Normal platelet thrombocytopenia are particularly important to detect because of the risk of developing onco-hematological pathology. The genetic origin of this category of thrombocytopenia has long been limited to mutations in the runx1 gene. More recently, mutations on the ankrd26 promoter have been reported. The work I did during my thesis helped to involve the etv6 gene in this group of thrombocytopenia. Concerning this gene six families have pathogenic mutations. All these mutations are the cause of a loss of the repressive activity of the gene and a high number of CD34+ cells circulating in the blood revealing the role of ETV6 in the onco-hematological predisposition. In addition, megakaryopoiesis has two main anomalies. They associate an increase in the number of megakaryocytic progenitor colonies with the formation of reduced proplatelets.Concerning the itga2b and itgb3 genes, 3 families were studied. These genes encode the αIIbβ3 integrin. Integrin αIIbβ3 is a platelet receptor for fibrinogen and Von Willebrand factor, and plays a crucial role in thrombosis and hemostasis

Glanzmann thrombasthenia (GT) is a recessively inherited bleeding disorder caused by quantitative and, or, qualitative deficiencies of the platelet integrin αIIbβ3, which binds fibrinogen to mediate platelet aggregation at sites of vascular injury. The α- and β-subunits of αIIbβ3 are encoded by ITGA2B and ITGB3 respectively, and many genetic defects resulting in reduced expression or dysfunction of αIIbβ3 have been described. A previous survey of UK GT patients that was carried out by our group identified 14 uncharacterised nonsynonymous alterations in ITGA2B and ITGB3 that predicted amino acid substitutions in different domains of αIIb and β3. This study used combined in silico and in vitro approaches to confirm the pathogenicity of 13 of these alterations; eight ITGB3 variants predicting p.Trp11Arg, p.Pro189Ser, p.Glu200Lys, p.Trp264Leu, p.Ser317Phe, p.Cys547Trp, p.Cys554Arg and p.Ile665Thr substitutions in β3 and five ITGA2B variants predicting p.Asp396Asn, p.Leu492Pro, p.Ile596Thr, p.Asn670Lys and p.Glu698Asp substitutions in αIIb. With the exception of the β3_p.Glu200Lys and αIIb_p.Glu698Asp variants, in silico analysis predicted all variants to be pathogenic. Compared to cells expressing wild-type (WT) αIIbβ3, there was an almost complete absence of surface αIIbβ3 in cells expressing the p.Trp11Arg, p.Pro189Ser, Trp264Leu and Ser317Phe β3 variants (p<0.0001). In contrast, the β3_p.IIe665Thr variant was expressed at similar levels to WT, but showed reduced ability to bind an antibody that is specific for the active conformation of the receptor, PAC1 (p<0.01) and also fibrinogen (p<0.05), while the β3_p.Glu200Lys variant does not appear to cause dysfunction to αIIbβ3. Interestingly, cells expressing the p.Cys547Trp and p.Cys554Arg β3 variants showed moderate reductions in surface expression of αIIbβ3 (p<0.0001) that exhibited spontaneous activation (p<0.0001;p<0.001). The majority of the substitutions in β3 resulted in greater than 90% reductions (p<0.0001) in membrane expression of αvβ3 with the exception of the p.Glu200Lys and p.Pro189Ser substitutions which resulted in 78% (p<0.0001) and 21% (p<0.001) reductions in membrane expression of αvβ3, respectively. There was a severe reduction in membrane αIIbβ3 on cells expressing the p.Asp396Asn, the p.Leu492Pro and the p.Ile596Thr αIIb variants (p<0.0001) while the p.Asn670Lys αIIb was expressed at similar levels to wild-type αIIb. Interestingly, the p.Leu492Pro and p.Asn670Lys variants showed reduced ability to bind PAC1 (p<0.0001;p<0.01) and fibrinogen (p<0.0001;p<0.01). The c.2094G>T transversion in ITGA2B, predicted to cause a p.Glu698Asp substitution in αIIb, was associated with a ii splicing defect, resulting in the deletion of exon 20 from the ITGA2B RNA , and the loss of αIIbβ3 expression. These findings confirm the pathogenicity and demonstrate the underlying mechanisms associated with GT for the majority of the variants studied. Interestingly, while the β3_p.Glu200Lys variant does not appear to cause αIIbβ3 dysfunction, it does result in a reduction in αvβ3 (vitronectin) receptor expression, though it remains unknown how this defect results in GT.

La thrombasthénie de Glanzmann (TG) est une maladie autosomique récessive liée à undéficit quantitatif et/ou qualitatif de l’intégrine αIIbβ3, principale glycoprotéine présente à la surfacedes plaquettes. Ce complexe sert de récepteur au fibrinogène plasmatique, permettant ainsi auxplaquettes de s’agréger entre-elles. Notre étude visait à caractériser les anomalies génétiquesresponsables de TG chez 76 familles d’origine différente. Les signes hémorragiques présentés parles patients étaient principalement des épistaxis, des pétéchies, des saignements gastro-intestinauxou des ménorragies. Les mutations présentes dans les gènes ITGA2B ou ITGB3 ont été identifiéespar séquençage direct. Tous les exons, ainsi que les régions introniques flanquantes, ont étéétudiées, permettant ainsi d’identifier 78 variations génétiques, dont 57 n’avaient jamais étérapportées. Des mutations tronquantes ou de l’épissage étaient présentes dans près de la moitié descas. Les mutations faux-sens représentaient également une forte proportion des anomaliesmoléculaires retrouvées (50% environ). Le caractère délétère de ces mutations a été confirmé parl’utilisation de méthodes in silico et/ou in vitro, permettant de caractériser les domaines essentiels àla structure et à la fonction des sous-unités αIIb et β3. En termes de corrélation génotype-phénotype,notre étude n’a pas permis de mettre en évidence d’association claire entre certaines mutations et lesyndrome hémorragique présenté par les patients. Cependant, ce travail permet de mieuxcomprendre les mécanismes impliqués dans la structure et le fonctionnement de la principaleintégrine plaquettaire
Glanzmann thrombasthenia (GT) is a rare autosomal recessive disorder characterized byquantitative and/or qualitative defect of the platelet αIIbβ3 integrin. Naturally occurring mutations inITGA2B or ITGB3 genes are responsible for the disease. Sanger sequencing analysis was applied tomutation screening of 83 diagnosed GT patients. 78 different sequence variations were identified ofwhich 57 had never been previously described. Among the novel identified mutations, truncative,missense and splice site mutations were observed. Therefore, we have identified a spectrum ofunreported mutations that may be of value to decipher the role of specific regions within αIIbβ3

La tesi està composta per dos articles publicats amb data de 12 de desembre de 2017 i 26 d’agost de 2020. Tots dos articles han estat admesos per formar part d’aquesta tesi en el seu format de compendi d’articles per la Comissió Acadèmica de el Programa de Doctorat de Cirurgia i Ciències Morfològiques, en la línia d’investigació d’Anatomia Patològica, amb data de 28 de juliol de 2020. El treball aquí presentat està dividit en diferents apartats; el primer d’ells consta d’una introducció conjunta on es presenten els antecedents i coneixements previs en els quals es basen les hipòtesis i els objectius exposats tot seguit. A continuació, l’apartat de resultats està dividit en dos grans capítols on en cada un d’ells es despleguen els articles que formen la tesi. En el primer capítol es presenta un estudi amb el qual es pretenia identificar factors que a través de la seva regulació traduccional, sota condicions d’estrès hipòxic i de bloqueig de la via canònica de la síntesi proteica, fossin capaços de conferir resistència a aquestes condicions. Per a això es va dur a terme un RNA-Seq d’ARN polisòmic en condicions de: 1) normòxia, 2) hipòxia (0,5% d’oxigen), 24h., 3) normòxia + PP242 i 4) hipòxia + PP242, un conegut inhibidor de mTORC1 / mTORC2. La Integrina Beta 3 (ITGB3) va ser un dels candidats obtinguts. Posteriors estudis van demostrar que la seva inhibició, i de manera més categòrica en hipòxia, no només va augmentar l’apoptosi i va reduir la supervivència i la migració cel·lular, sinó que ITGB3 era requerida per a l’activació sostinguda de la via de TGF-β. Tot això sumat al fet que la inhibició de ITGB3 va reduir significativament la metàstasi pulmonar i va millorar la supervivència general en ratolins. Cal destacar, que en el primer capítol s’ha afegit un sub-apartat de dades no publicades. Aquests resultats es van obtenir durant la meva estada de tres mesos al laboratori del Professor Alan McIntyre a la Universitat de Nottingham, Anglaterra. Durant aquesta estada, l’article va ser acceptat per la revista i per aquest motiu els resultats obtinguts no van ser afegits a la publicació. El capítol II està centrat en el paper de ITGB3 en la comunicació intercel·lular a través de vesícules extracel·lulars i com la desregulació d’aquesta podria explicar la disminució en l’aparició de metàstasis en cèl·lules amb la integrina inhibida descrita en el capítol I. En aquest estudi, hem definit un paper essencial i fins ara desconegut de ITGB3 en l’absorció de vesícules. El mecanisme funcional que està basat la interacció de ITGB3 amb glucoproteïnes heparan sulfatades (HSPG) i en el procés de reciclatge de les integrines, també ha sigut descrit. La combinació d’ambdós mecanismes permet la captura de vesícules extracel·lulars i la seva internalització mitjançada per endocitosi. A més, en el complex d’internalització són molt importants altres proteïnes com la GTPasa Dinamina i la quinasa d’adhesions focals (FAK). Les dues proteïnes són clau per dur a terme l’endocitosi i la posterior entrada de les vesícules extracel·lulars a la ruta endocítica a través dels endosomes. Conseqüentment, podem afirmar que ITGB3 té un paper central en la comunicació intracel·lular a través de vesícules extracel·lulars, mecanisme que es pressuposa crític durant la metàstasi tumoral. Després de l’apartat de resultats, es continua amb una discussió conjunta dels dos capítols on a més de discutir sobre els resultats obtinguts, es debaten les possibles aplicacions translacionals, majoritàriament amb un enfocament terapèutic. Per acabar, s’exposen les conclusions més rellevants extretes d’aquesta tesi.
La tesis está compuesta por dos artículos, publicados a fecha de 12 de diciembre de 2017 y 26 de agosto de 2020. Ambos artículos han sido admitidos para formar parte de dicha tesis en su formato de compendio de artículos por la Comisión Académica del Programa de Doctorado de Cirugía y Ciencias Morfológicas, en la línea de investigación de Anatomía Patológica, con fecha de 28 de julio de 2020. El trabajo aquí presentado está dividido en diferentes apartados; el primero de ellos consta de una introducción conjunta donde se presentan los antecedentes y conocimientos previos en los cuales se basan las hipótesis y los objetivos expuestos seguidamente. A continuación, el apartado de resultados está dividido en dos grandes capítulos donde en cada uno de ellos se despliegan los artículos que forman la tesis. En el primer capítulo se presenta un estudio con el que se pretendía identificar factores que a través de su regulación traduccional, bajo condiciones de estrés hipóxico y de bloqueo de la vía canónica de la síntesis proteica, fueran capaces de conferir resistencia a dichas condiciones. Para ello se llevó a cabo un RNA-Seq de ARN polisómico en condiciones de: 1) normoxia, 2) hipoxia (0,5% de oxígeno), 24h., 3) normoxia + PP242 e 4) hipoxia + PP242, un conocido inhibidor de mTORC1/mTORC2. La Integrina Beta 3 (ITGB3) fue uno de los candidatos obtenidos. Posteriores estudios demostraron que su inhibición, y de manera más categórica en hipoxia, no solo aumentó la apoptosis y redujo la supervivencia y la migración celular, si no que ITGB3 era requerida para la activación sostenida de la vía de TGF-β. Todo ello sumado a que la inhibición de ITGB3 redujo significativamente la metástasis pulmonar y mejoró la supervivencia general en ratones. Cabe destacar, que en el primer capítulo se ha añadido un sub-apartado de datos no publicados. Estos resultados se obtuvieron durante mi estancia de tres meses en el laboratorio del Profesor Alan McIntyre en la Universidad de Nottingham, Inglaterra. Durante dicha estancia, el artículo fue aceptado por la revista y por este motivo los resultados obtenidos no fueron añadidos a la publicación. El capítulo II está centrado en el rol de ITGB3 en la comunicación intercelular a través de vesículas extracelulares y como la desregulación de ésta podría explicar la disminución en la aparición de metástasis en células con la integrina inhibida descrita en el capítulo I. En este estudio, hemos definido un rol esencial y hasta ahora desconocido de ITGB3 en la absorción de vesículas. El mecanismo funcional que está basado en la interacción de ITGB3 con glucoproteínas heparán sulfatadas (HSPG) y en el proceso de reciclaje de las integrinas también ha sido descrito. La combinación de ambos mecanismos permite la captura de vesículas extracelulares y su internalización mediada por endocitosis. Además, en el complejo de internalización juegan un papel importante otras proteínas como la GTPasa dinamina y la quinasa de adhesiones focales (FAK). Ambas proteínas son clave para llevar a cabo la endocitosis y la posterior entrada de las vesículas extracelulares a la ruta endocítica a través de los endosomas. Consecuentemente, podemos afirmar que ITGB3 tiene un papel central en la comunicación intracelular a través de vesículas extracelulares, mecanismo que se presupone crítico durante la metástasis tumoral. Tras el apartado de resultados, se continúa con una discusión conjunta de ambos capítulos donde además de discutir sobre los resultados obtenidos, se debaten las posibles aplicaciones traslaciones, mayoritariamente con un enfoque terapéutico, de dichos estudios. Para acabar, se exponen las conclusiones más relevantes extraídas de dicha tesis.
The thesis is made up of two articles dated December 12, 2017 and August 26, 2020. Both articles have been admitted to form part of the said thesis in its compendium format of articles by the Academic Commission of the Doctorate Program of Surgery and Morphological Sciences, in the research line of Pathological Anatomy, dated July 28th, 2020. The work presented here is divided into different sections. The first of them consists of a joint introduction where the background and prior knowledge on which the hypotheses and the objectives set forth below are based are presented. Next, the results section is divided into two large chapters where the publications that make up this thesis are displayed. The first chapter refers to the first article already published which aimed to identify factors that through their translational regulation under hypoxic stress conditions and the blockage of the canonical pathway of protein synthesis, were able to confer survival resistance to these conditions. For this, RNA-Seq of polysomal RNA was carried out under conditions of 1) normoxia, 2) hypoxia (0.5% oxygen), 24h., 3) normoxia + PP242, and 4) hypoxia + PP242, a known mTORC1 / mTORC2 inhibitor. Integrin Beta 3 (ITGB3) was one of the obtained candidates. Subsequent studies demonstrated that its inhibition, and more categorically in hypoxia, not only increased apoptosis and reduced survival and cell migration, but that ITGB3 was required for sustained activation of the TGF-β pathway. All of this added to the fact that ITGB3 inhibition significantly reduced lung metastasis and improved overall survival in mice. It should be noted that in the first chapter a sub-section of unpublished data has been added. These results were obtained during my three-month stay in Professor Alan McIntyre’s laboratory at the University of Nottingham, England. During this stay, the article was accepted by the magazine, and for this reason, the obtained results were not added to the publication. Chapter II focuses on the role of ITGB3 in extracellular vesicle-based intercellular communication and how this could explain the decrease in the formation of metastasis in cells with ITGB3 inhibition, described in Chapter I. In this study, we have described an essential and far unknown role of ITGB3 in the absorption of vesicles. The functional requirement is based on the interaction of ITGB3 with Heparan Sulfated Glycoproteins (HSPG) and with the recycling process of the integrins. The combination of both mechanisms allows the capture of extracellular vesicles and their internalization mediated by endocytosis. Also, other proteins such as the GTPase Dynamin and Focal Adhesion Kinase (FAK) play an important role in the internalization complex. Both proteins are key to carrying out endocytosis and the subsequent entry of extracellular vesicles into the endosomes of the endocytic pathway. Thus, ITGB3 has a central role in intracellular communication via extracellular vesicles, proposed to be critical for cancer metastasis. After the results section, a combined discussion of both chapters is presented, where, in addition to discussing the obtained results, we discuss the possible translational applications, mostly with a therapeutic approach. To finish, the most relevant conclusions drawn are exposed.

Introdução: O diabetes mellitus tipo 2 (DM2) representa cerca de 90% dos tipos de diabetes e vem atingindo de forma cada vez mais intensa a população adulta e atualmente também jovens e até crianças. Uma dieta hipercalórica, aliada ao sedentarismo tem sido junto com a predisposição genética os principais desencadeantes das complicações crônicas associadas com o DM2. Infelizmente, são essas complicações que levam ao grande aumento da morbidade e mortalidade que pode chegar até 80% nesta doença, sendo que as principais são as de natureza micro e macrovascular. Complicações microvasculares compreendem a retinopatia diabética (RD), a nefropatia diabética (ND) e a neuropatia periférica (NP), enquanto que as complicações macrovasculares compreendem a doença cardiovascular (DCV), a doença arterial periférica (DAP) e o acidente vascular cerebral (AVC). Pacientes com DM2 em sua fase inicial já podem apresentar um quadro protrombótico que só tende a piorar com a progressão da doença. Esse quadro protrombótico é resultante principalmente do processo inflamatório e da disfunção endotelial. Polimorfismos genéticos relacionados com as diferentes fases da hemostasia podem contribuir para o aumento ou diminuição no risco de formação de trombos arteriais e venosos que podem afetar a micro e macrovasculatura destes indivíduos. Objetivos: Investigar a influência de polimorfismos envolvidos com a hemostasia como fatores de risco para o desenvolvimento de complicações crônicas micro e macrovasculares em pacientes com DM2. Metodologia: Foi realizado um estudo de caso controle aninhado em uma coorte de pacientes DM2 não relacionados provenientes de um estudo multicêntrico no sul do Brasil. Os pacientes DM2 foram divididos em 2 grupos de estudo. Para o grupo com complicação macrovascular estudou-se 404 pacientes DM2. Casos para a complicação macrovascular foram definidos como tendo cardiopatia isquêmica (CI), acidente vascular cerebral isquêmico (AVCI) ou DAP enquanto que controles foram definidos como pacientes com pelo menos 5 anos de DM2 e sem a respectiva complicação. Para o estudo das complicações microvasculares 393 pacientes com DM2 foram estudados. Os casos foram definidos como tendo RD, ND ou neuropatia sensório distal (NSD). Controles para a complicação microvascular foram pacientes com pelo menos 10 anos de DM2 e sem a respectiva complicação. Os polimorfismos estudados foram testados em duplicata utilizando-se a PCR seguida de RFLP quando necessário. Foram investigados nove polimorfismos assim distribuídos: Na fase da coagulação foram estudados cinco polimorfismos (FGB rs1800790, F2 rs1799963, FV rs6025 F7 rs5742910 e F13A rs5985); dois (PLAT rs4646972 e PAI-1 rs1799768) na fase da fibrinólise e um (ITGB3 rs5918) na fase plaquetária. O polimorfismo da MTHFR rs1801133 envolvido com a hiperhomocisteínemia é considerado um fator de risco para DCV e por isso foi incluído. Para a análise estatística foi utilizado o teste do χ2 para a comparação das frequências genotípicas e alélicas. Os polimorfismos com diferença significativa foram testados na regressão de Poisson com variância robusta ajustado pelas variáveis de confusão. Para as complicações microvasculares também foi utilizado o teste do χ2 com análise de resíduo ajustado. Resultados: O polimorfismo do receptor plaquetário ITGB3 rs5918 apresentou associação com os desfechos AVCI e DAP. Para o desfecho AVCI o genótipo 176TC mostrou associação significativa [(PR = 2.04(1.11-3.73); P = 0.021], enquanto que para a DAP a associação foi com o genótipo 176CC [PR = 1.90(1.29-2.81); P = 0.001]. Em relação às complicações microvasculares o único polimorfismo que mostrou associação foi o PAI-1 rs1799768. Neste caso, o polimorfismo demonstrou ter uma associação inesperada para o alelo 4G como um fator de proteção quando comparamos pacientes com e sem ND [PR = 0.71(0.57-0.89); P = 0.003]. Porém, quando foi estratificado o grupo de pacientes com ND de acordo com a severidade, foi possível demonstrar usando a análise de resíduo ajustado do teste do χ2 que havia uma diminuição significativa na frequência do alelo 4G somente no estágio mais avançado da doença renal (P = 0.009; AR = -2.95) o que sugere o seu envolvimento com uma maior taxa de mortalidade na ND. Também foi possível mostrar que o alelo de risco 4G está significativamente associado com a cardiopatia isquêmica nos indivíduos com ND (P = 0.03; AR = 2.5). Conclusões: Os pacientes com DM2 portadores do alelo de risco 176C do polimorfismo ITGB3 rs5918 apresentam um risco significativamente aumentado de desenvolver AVCI e DAP, enquanto que os portadores do alelo de risco 4G do polimorfismo PAI-1 rs1799768 provavelmente apresentem maior risco de desenvolver ND. Além disso, os portadores do alelo 4G e que tem ND apresentaram um risco significativamente aumentado de desenvolverem CI.
Introduction: Type 2 diabetes mellitus (T2DM) represents approximately 90% of the diabetes types, increasingly affecting the adult population and nowadays also occurring in young adults and children. Hypercaloric diets, sedentarism and genetic predisposition are the main triggering factors of chronic complications associated to T2DM. Unfortunately, these are the complications that lead to a considerable increase in morbidity and mortality, which may reach 80%, with the main complications being of micro- and macrovascular nature. The microvascular complications are diabetic retinopathy (DR), diabetic nephropathy (DN) and peripheral neuropathy (PN). The macrovascular complications include cardiovascular disease (CVD), peripheral arterial disease (PAD) and stroke. In the initial T2DM stage, patients may present a prothrombotic state that tends to worsen as the disease evolves. This prothrombotic state results mainly from the inflammatory process and from the endothelial dysfunction. Genetic polymorphisms related to the different stages of hemostasis may play a role in the increase or decrease of the risk of arterial and venous thrombus, which may affect the micro- and the macrovasculature of these individuals. Objectives: To investigate the influence of the polymorphisms related to hemostasis as risk factors for the development of micro- and macrovascular complications in T2DM patients. Methods: A nested case-control study was conducted with a cohort of unrelated T2DM patients from a multicenter study made in southern Brazil. T2DM patients were divided in two groups. The macrovascular complication group included 404 T2DM patients. Macrovascular complications were defined according to the presence of the following criteria: ischemic heart disease (IHD), ischemic stroke (IS) or PAD. The control group was formed by patients who had had T2DM for at least five years but without the respective complications. The microvascular complication group included 393 T2DM patients. Microvascular complications were defined based on the presence of the following criteria: DR, DN, or distal sensory neuropathy (DSN). The controls used in the investigation of the microvascular complications were patients who had T2DM for at least 10 years, without the respective complications. The polymorphisms investigated were analyzed by PCR with RFLP, when necessary. In total, nine polymorphisms were studied. Five polymorphisms (FGB rs1800790, F2 rs1799963, FV rs6025, F7 rs5742910 and F13A1 rs5985) were investigated for the coagulation stage, two (PLAT rs4646972 and PAI-1 rs1799768) for the fibrinolysis stage, and one (ITGB3 rs5918) for the platelet stage. The polymorphism MTHFR rs1801133, associated to hyperhomocysteinemia, which is considered a risk factor for IHD, was also investigated. The statistical analysis used the χ² test to compare genotypic and allelic frequencies. The polymorphisms presenting significant differences were tested using the Poisson regression with robust variance adjusted for the confounding variables. The χ² test with the analysis of adjusted residues was also used for microvascular complications. Results: The polymorphism of the platelet receptor ITGB3 rs5918 was associated with the outcomes IS and PAD. Considering IS, the genotype 176TC exhibited significant association [(PR = 2.04(1.11-3.73); P = 0.021], while considering PAD the association was with genotype 176CC [PR = 1.90(1.29-2.81); P = 0.001]. Regarding the microvascular complications, the only polymorphism that presented association was PAI-1 rs1799768. In this case, the polymorphism demonstrated an unexpected association with allele 4G as a protection factor when patients with and without DN [PR = 0.71(0.57-0.89); P = 0.003]. However, when the group of patients with DN was stratified in terms of severity, it was possible to demonstrate a significant decrease in 4G allele frequency only n the more advanced stage of the renal disease, using the adjusted residue of the χ2 test (P = 0.009; AR = -2.95), which suggests its involvement with a higher mortality rate in DN. It was also possible to show that the risk allele 4G is significantly associated with ischemic cardiopathy in individuals with DN (P = 0.03; AR = 2.5). Conclusions: The T2DM patients carriers of the risk allele 176C of the polymorphism ITGB3 rs5918 present a significantly increased risk of developing IS and PAD, while carriers of the risk allele 4G of the polymorphism PAI-1 rs1799768 probably present higher risk of developing DN. Apart from this, this group of subjects also presented a significant risk of developing IHD.

BACKGROUND:Airway hyperresponsiveness (AHR), a primary characteristic of asthma, involves increased airway smooth muscle contractility in response to certain exposures. We sought to determine whether common genetic variants were associated with AHR severity.METHODS:A genome-wide association study (GWAS) of AHR, quantified as the natural log of the dosage of methacholine causing a 20% drop in FEV1, was performed with 994 non-Hispanic white asthmatic subjects from three drug clinical trials: CAMP, CARE, and ACRN. Genotyping was performed on Affymetrix 6.0 arrays, and imputed data based on HapMap Phase 2, was used to measure the association of SNPs with AHR using a linear regression model. Replication of primary findings was attempted in 650 white subjects from DAG, and 3,354 white subjects from LHS. Evidence that the top SNPs were eQTL of their respective genes was sought using expression data available for 419 white CAMP subjects.RESULTS:The top primary GWAS associations were in rs848788 (P-value 7.2E-07) and rs6731443 (P-value 2.5E-06), located within the ITGB5 and AGFG1 genes, respectively. The AGFG1 result replicated at a nominally significant level in one independent population (LHS P-value 0.012), and the SNP had a nominally significant unadjusted P-value (0.0067) for being an eQTL of AGFG1.CONCLUSIONS:Based on current knowledge of ITGB5 and AGFG1, our results suggest that variants within these genes may be involved in modulating AHR. Future functional studies are required to confirm that our associations represent true biologically significant findings.

Les tumeurs solides emploient diverses stratégies afin de se maintenir dans l’organisme et d’échapper à l’inhibition du système immunitaire. Un des mécanismes les plus puissants est la production de la cytokine Transforming Growth Factor Beta (TGF-bêta). Cependant, cette cytokine est sécrétée dans le micro-environnement tumoral sous une forme inactive, incapable de se lier à son récepteur et donc d’exercer ses fonctions hautement immunosuppressives. Ces travaux de thèse démontrent qu’une population de lymphocytes T (LT) CD4+ dite T régulateurs (Tregs), qui exprime le facteur de transcription Forkhead box P3 (Foxp3), est responsable de l’activation du TGF-bêta au sein de la tumeur. Nous avons montré que parmi les cellules du système immunitaire, les Tregs constituent la principale population exprimant l’intégrine avb8 (Itgb8), protéine responsable de l’activation du TGF-bêta. L’absence de l’Itgb8 spécifiquement à la surface des Tregs entraîne une forte diminution de la croissance tumorale. Par conséquent, l’activation de la signalisation du TGF-bêta est réduite dans les LT CD8+ qui infiltrent la tumeur, conduisant à une exacerbation de leurs fonctions cytotoxiques et donc à une élimination accrue des cellules tumorales. La relevance de ces données obtenues chez la souris a été confirmée chez l’Homme à la fois par des approches ex vivo sur des tumeurs fraîches ainsi que par des approches bio-informatiques et biostatistiques à partir d’étude de cohortes de patients. Nous proposons donc que les Tregs et les cellules tumorales travaillent de concert pour fournir une source bio-active de TGF-bêta capable de réprimer efficacement la réponse immunitaire anti-tumorale et donc de permettre à la tumeur d’échapper au système immunitaire
Solid tumors employ diverse strategies to be maintained in the organism and escape the suppression mediated by the immune system. One of the most powerful mechanisms they use is through the production of Transforming Growth Factor Beta (TGF-beta). However, this cytokine is secreted within the tumor microenvironment in its inactive form, unable to bind to its receptor and exert its highly immunosuppressive functions. The present thesis project demonstrates that a population of CD4+ T lymphocytes called regulatory T cells (Tregs), which express the transcription factor Forkhead box P3 (Foxp3), is responsible for TGF-beta activation in tumors. We show that among the cells of the immune system, Tregs constitute the main population expressing the integrin avb8 (Itgb8) which is responsible for TGF-beta activation. The absence of Itgb8 specifically on Tregs surface leads to strong decrease of tumor growth. As a result, TGF-beta signaling pathway is impaired in tumor infiltrating CD8+ T lymphocytes leading to exacerbation of their cytotoxic and efficient elimination of tumor cells. The relevance of these data obtained in mice was confirmed in the human pathology by ex vivo approaches using fresh tumors as well as by bioinformatics and biostatistics approaches from studies on patient cohorts. We propose that Tregs and tumor cells cooperate to provide a bioactive source of TGF-beta which is able to efficiently repress the anti-tumor response and thus allowing tumors to escape the immune system

碩士
中國醫藥大學
癌症生物學研究所碩士班
101
In past statistics, metastasis of cancer is the main factor of death. In previous study demonstrate that knockdown of ADAM9 could inhibit migration, invasion and metastasis of prostate cancer. Our Lab confirmed that interaction between ADAM9 and hemidesmosome components by mass spectrometry, in addition, ADAM9 promotes hemidesmosome disassemble. The central theme of our study is that during cancer cell migration and invasion, cancer cell need to loosen from anchored region which is regulated by hemidesmosome for the protrusion of lamellipodia. Once lamellipodia protrusion completed, cell migration front need to re-anchored on matrix to secure the protrusion. This may need the new formation of either integrin or hemidesmosome component. In addition, retraction of tailing will also need the loosen of integrin α6β4 and hemidesmosome from tail. This may need the assisting by ADAM9 for helping integrin β4 endocytosis and degradation. Therefore, we observed endocytosis of integrin β4 by immunofluorescence staining and analyzed degradation of integrin by cycloheximide pulse chase assay. Further observed pathway after endocytosis of integrin β4 by Rab GTPases as endosomal trafficking pathway markers. In conclusion, these results showed that ADAM9 promotes velocity of endocytosis and degradation of integrin β4. Moreover, laminin inhibits endocytosis and degradation of integrin β4. We also confirm integrin β4 entering into LAMP1 enriched vesicle for lysosomal degradation by Rab7 as lysosome degradation marker. Our results suggested that ADAM9 and laminin could be a useful agent for prostate cancer.