Adhesion to fibronectin via α5ß1 integrin supports expansion of the megakaryocyte lineage in primary myelofibrosis.

Excessive accumulation of extracellular matrix (ECM) is a hallmark of bone marrow (BM) milieu in primary myelofibrosis (PMF). Because cells have the ability to adhere to the surrounding ECM through integrin receptors, we examined the hypothesis that an abnormal ECM-integrin receptor axis contributes to BM megakaryocytosis in JAK2V617F+ PMF. Secretion of ECM protein fibronectin (FN) by BM stromal cells from PMF patients correlates with fibrosis and disease severity. Here, we show that Vav1-hJAK2V617F transgenic mice (JAK2V617F+) have high BM FN content associated with megakaryocytosis and fibrosis. Further, megakaryocytes from JAK2V617F+ mice have increased cell surface expression of the α5 subunit of the α5ß1 integrin, the major FN receptor in megakaryocytes, and augmented adhesion to FN compared with wild-type controls. Reducing adhesion to FN by an inhibitory antibody to the α5 subunit effectively reduces the percentage of CD41+ JAK2V617F+ megakaryocytes in vitro and in vivo. Corroborating our findings in mice, JAK2V617F+ megakaryocytes from patients showed elevated expression of α5 subunit, and a neutralizing antibody to α5 subunit reduced adhesion to FN and megakaryocyte number derived from CD34+ cells. Our findings reveal a previously unappreciated contribution of FN-α5ß1 integrin to megakaryocytosis in JAK2V617F+ PMF.

Titanium with nanotopography induces osteoblast differentiation through regulation of integrin αV.

Topographical modifications of titanium (Ti) at the nanoscale level generate surfaces that regulate several signaling pathways and cellular functions, which may affect the process of osseointegration. Here, we investigated the participation of integrin αV in the osteogenic capacity of Ti with nanotopography. Machined titanium discs (untreated) were submitted to treatment with H2 SO4 /H2 O2 to produce the nanotopography (nanostructured). First, the greater osteogenic capacity of the nanotopography that increased osteoblast differentiation of mesenchymal stem cells compared with untreated topography was shown. Also, the nanostructured surface increased (regulation ≥ 1.9-fold) the gene expression of 6 integrins from a custom array plate utilized to evaluate the gene expression of 84 genes correlated with cell adhesion signaling pathway, including integrin αV, which is involved in osteoblast differentiation. By silencing integrin αV in MC3T3-E1 cells cultured on nanotopography, the impairment of osteoblast differentiation induced by this surface was observed. In conclusion, it was shown that nanotopography regulates the expression of several components of the cell adhesion signaling pathway and its higher osteogenic potential is, at least in part, due to its ability to upregulate the expression of integrin αV. Together with previous data that showed the participation of integrins α1, ß1, and ß3 in the nanotopography osseoinduction activity, we have uncovered the pivotal role of this family of membrane receptors in the osteogenic potential of this surface.

Molecular analysis of biocompatibility of anodized titanium with deposited silver nanodendrites.

Titanium (>99.6% purity) and its anodically oxidized modifications, with and without deposited silver nanodendrites regarding its biocompatibility were evaluated. In human gingival fibroblasts and osteoblast cell lines grown on tested samples, the level of expression of genes encoding αV (ITGAV) and ß1 (ITGB1) integrin subunits also genes encoding focal adhesion (FAK) and extracellular-signal regulated (ERK) kinases was assessed. For this purpose, the qualitative and quantitative PCR technique was used. The expression of studied genes was dependent on the origin of cell lines and the type of evaluated material. The high expression of PBGD and ITGAV genes in fibroblasts grown on the surface of anodically modified titanium with deposited silver nanodendrites indicates potentially high biocompatibility of these samples for soft tissue cells. The high expression of the ITGB1 and ERK1 genes and the enhanced expression of the FAK gene in osteoblasts cells grown on the tested material was also observed. Summarizing, the nanocrystalline Ti modified with silver deposits showed higher biocompatibility in comparison with the conventional pure Ti samples.

Down regulation of ITGA4 and ITGA5 genes after formation of 3D spherules by human Wharton's jelly stem cells (hWJSCs).

Human Wharton's jelly mesenchymal stem cells (hWJSCs) are multipotent stem cells that could be aggregated into 3D spherules. ITGA4 and ITGA5 genes encode α4 and α5 subunits of integrins, respectively. In this study, we analyzed expression levels of ITGA4 and ITGA5 gene mRNAs in undifferentiated and 3D spherules forming hWJSCs in order to determine their expression pattern for possible future treatment of cancer cells in a co-culture fashion. For the purpose of obtaining hWJSCs, umbilical cords were collected from patients with caesarian section at full term delivery. The cells were then characterized according to cell surface markers using flow cytometry. Furthermore pluripotency of the obtained cells was verified. Subsequently the cells were aggregated in 3D spherules using hanging drop cultures. Expression levels of ITGA4 and ITGA5 gene mRNAs were determined by RT-PCR and Real time PCR, both in the initial undifferentiated cells and those aggregated in the spherules. The obtained hWJSCs demonstrated pluripotency, differentiating to adipogenic and osteogenic cells. They also expressed mesenchymal stem cell surface markers. Following the aggregation of these cells and formation of 3D spherules, mRNA expression levels of both genes were significantly reduced (P < 0.05) compared with the initial undifferentiated state. The results of this study demonstrated that aggregation of hWJSCs into spherules alters their expression of ITGA4 and ITGA5. The implications of such an alteration would require further research.

miR-25 Mediates Retinal Degeneration Via Inhibiting ITGAV and PEDF in Rat.

BACKGROUND: Age-related macular degeneration (AMD) is the main cause of irreversible blindness in the elderly. Oxidative stress in retinal pigment epithelium (RPE) is deemed to play a pivotal role in the pathogenesis of AMD. miR-25 functions as an essential modulator in response to oxidative-stress in several cell types, but its function in RPE cells is poorly understood. OBJECTIVE: To explore the roles of miR-25 in RPE cells and in the development of AMD. METHODS: A rat model of retinal degeneration was induced by sodium iodate (SI). Subretinal injection of antagomiR-25 was performed for the intervention while the scramble as control. Visual responses were recorded with Electroretinogram (ERG). TUNEL assay was performed to detect apoptosis. Phagosome quantification in vivo was performed to evaluate RPE cell function. Oxygen-glucose deprivation treatment was performed to mimic in vitro oxidative stress. Gene expression at mRNA level and protein level were performed by quantitative polymerase chain reaction (qPCR) and Western Blot, respectively. The pigment epithelium derived factor (PEDF) level in the cultured medium was measured by Enzyme-linked immunosorbent assay (ELISA). The interaction between miR-25 and integrin αV (IGTAV) / PEDF 3'UTR was examined by dual luciferase assay. Chromatin immunoprecipitation (ChIP) assay was performed to examine its transcriptional regulation of miR-25. RESULTS: Oxidative stress up-regulated miR-25 in RPE cells in very early stage, accompanied by decreased phagocytosis and reduced growth factor secretion in those cells. Such changes preceded RPE cell apoptosis and visual impairment in the SItreated rats. Furthermore, antagomiR-25 intervention effectively rescued RPE cells from degeneration in such model. The increased miR-25 was confirmed to mediate RPE degeneration through direct targeting IGTAV and PEDF. On the other hand, upstream, miR-25 was found to be up-regulated by STAT3 signaling under oxidative stress in both in vivo and in vitro models. CONCLUSION: Our findings demonstrate that, in SI-treated rats, oxidative stress activates STAT3 signaling which up-regulates miR-25 expression, in a very early stage. The increased miR-25 then inhibits ITGAV and PEDF expressions, resulting in RPE phagocytosis dysfunction and then RPE apoptosis and visual impairment as observed in patients with AMD. These findings lead us to a better understanding of AMD pathogenesis, and suggest that miR-25 could be a potential therapeutic target for oxidative stress related RPE diseases, like AMD.

Neuron-autonomous transcriptome changes upon ischemia/reperfusion injury.

Ischemic stroke and the following reperfusion, an acute therapeutic intervention, can cause irreversible brain damages. However, the underlying pathological mechanisms are still under investigation. To obtain a comprehensive, real-time view of the cell-autonomous mechanisms involved in ischemic stroke and reperfusion, we applied the next-generation sequencing (NGS) technology to characterize the temporal changes in gene expression profiles using primarily cultured hippocampal neurons under an oxygen-glucose deprivation/reperfusion (OGD/R) condition. We first identified the differentially expressed genes (DEGs) between normal cultured neurons, neurons with OGD, and neurons with OGD followed by reperfusion for 6 h, 12 h, and 18 h, respectively. We then performed bioinformatics analyses, including gene ontological (GO) and pathway analysis and co-expression network analysis to screen for novel key pathways and genes involved in the pathology of OGD/R. After we confirmed the changes of selected key genes in hippocampal cultures with OGD/R, we further validated their expression changes in an in vivo ischemic stroke model (MCAO). Finally, we demonstrated that prevention of the up-regulation of a key gene (Itga5) associated with OGD/R promoted hippocampal neuronal survival. Our research thereby provided novel insights into the molecular mechanisms in ischemic stroke pathophysiology and potential targets for therapeutic intervention after ischemic stroke.

Fibroblast paracrine TNF-α signaling elevates integrin A5 expression in idiopathic pulmonary fibrosis (IPF).

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a poor prognosis. Inflammatory cytokines play a significant role in IPF pathology. However, the fibroblast itself is also believed to be the primary effector in IPF. We hypothesized that the fibroblasts themselves secrete pro-inflammatory cytokines that could propagate IPF by affecting normal neighboring cells. Thus, we explored the effects of IPF fibroblast derived media on normal fibroblast characteristics. METHODS: Primary IPF/normal tissue derived fibroblast cultures were established and their supernatants were collected (IPF/N-SN, respectively). These supernatants were added to normal fibroblasts. Cell death (caspase-3, western blot), proliferation, viability (WST-1), migration (scratch test) and cell detachment (crystal violet and fibronectin adhesion assays) were tested. 10 inflammatory cytokines were measured by ELISA-based quantitative array. Integrin α5 (ITGA5), pIκBα, p/total STAT3 levels were measured by western blot/IHC. TNF-α involvement was confirmed using Infliximab ®, anti-TNF-α mAb. RESULTS: The IPF-SN facilitated fibroblast cell detachment and reduced cell migration (p < 0.05). Nevertheless, these effects were reversed when cells were seeded on fibronectin. The exposure to the IPF-SN also elevated ITGA5 levels, the fibronectin receptor, in addition to NFκB pathway activation (pIκBα↑ 150%, p < 0.05). In accordance, IPF derived fibroblasts were found to express higher ITGA5 than the normal cells (44%↑, p < 0.05). ITGA5 was also expressed in the fibroblastic foci. The IPF-SN contained high TNF-α levels (3-fold, p < 0.05), and Infliximab pretreatment successfully reversed all the above observations. CONCLUSION: We suggest a possible mechanism in which IPF fibroblast secreted TNF-α modifies neighboring fibroblast cell behavior.

Integrin α5 promotes tumor progression and is an independent unfavorable prognostic factor in esophageal squamous cell carcinoma.

The integrin family plays a major role in complex biological events such as differentiation, development, wound healing, and the altered adhesive and invasive properties of tumor cells. The expression and function of integrin α5 in esophageal squamous cell carcinoma (ESCC) are not clear. Here, by using tissue microarrays and immunohistochemical method, integrin α5 expression was retrospectively evaluated in 147 samples of human ESCC. Results showed that expression of integrin α5 was heterogeneous and varied from negative to intense expression in a membrane and cytoplasmic distribution manner. High expression of integrin α5 was significantly correlated with lymph node metastasis (P = .042) and tumor size (P = .042). Kaplan-Meier analysis revealed that high expression of integrin α5 was related to poor overall survival of ESCC patients (P = .018). Multivariate analysis suggested that integrin α5 expression status was an independent prognostic factor for ESCC (P = .003). Moreover, integrin α5 expression was associated with the survival of patients with lymph node metastasis (P = .020), but did not influence the survival of patients without lymph node metastasis. Finally, we found that RNAi-mediated knockdown of integrin α5 led to decreased growth, migration, and invasion of ESCC cells. Combined, integrin α5 might play important roles in the progression of ESCC. Integrin α5 is a novel biomarker to predict the prognosis of ESCC patients.

Expression of α5-integrin, α7-integrin, Ε-cadherin, and N-cadherin in localized prostate cancer.

OBJECTIVE: To explore the correlation between the expression of α5-integrin, α7-integrin, Ε-cadherin, and N-cadherin in prostate cancer (PCa) and its clinicopathological data including tumor grade and clinical stage. METHODS: The expression of α5-integrin, α7-integrin, Ε-cadherin, and N-cadherin was examined in 157 cases of PCa and adjacent normal prostatic tissue by immunohistochemical assay, and the correlation with clinicopathological features was analyzed. RESULTS: Expressions of α5-integrin, α7-integrin, and Ε-cadherin in PCa were lower than those in normal prostatic tissues (P<0.05). N-cadherin expression was higher in cancer prostatic tissue than in normal prostatic tissues (P<0.05). The reduced expression of α5-integrin, α7-integrin, and Ε-cadherin was related to Gleason score, pathological stage, lymph node metastasis, and prostate-specific antigen level, but it was not associated with positive surgical margins and patient age. The increased expression of N-cadherin was related to Gleason score, pathological stage, lymph node metastasis, and prostate-specific antigen level, but not to age and positive surgical margins. The expression of E-cadherin was highly negatively correlated with that of N-cadherin and also positively correlated with that of α5-integrin and α7-integrin. CONCLUSION: The reduced expression of α5-integrin, α7-integrin, and Ε-cadherin and abnormal expression of N-cadherin play an important role in the occurrence and development of PCa. The results indicate that these have potential values in the diagnosis and are predictable indices in the proliferation of PCa.

Functional Impact of Collagens on the Activity Directed by the Promoter of the α5 Integrin Subunit Gene in Corneal Epithelial Cells.

PURPOSE: The early step of corneal wound healing is characterized by the massive production of fibronectin (FN), whose secretion is progressively replaced by collagens from the basal membrane as wound healing proceeds. Here, we examined whether expression of the gene encoding the α5 subunit from the FN-binding integrin α5ß1 changes as corneal epithelial cells (CECs) are cultured in the presence of collagen type I (CI) or type IV (CIV). METHODS: Responsiveness of the α5 gene toward collagen was determined by transfection of α5 promoter/chloramphenicol acetyltransferase (CAT) plasmids into rabbit and human CECs cultured on BSA or collagens. Electrophoretic mobility shift assays and Western blots were used to monitor the transcription factors required for basal α5 gene transcription in the presence of collagens. Gene profiling on microarrays was used to determine the impact of collagens on the patterns of genes expressed by CECs. RESULTS: All collagen types repressed the full-length α5/CAT promoter activity in confluent CECs. A moderate increase was observed in subconfluent rabbit CECs grown on CIV but not on CI. These collagen-dependent regulatory influences also correlated with alterations in the transcription factors Sp1/Sp3, NFI, and AP-1 that ensure α5 gene basal transcription. Microarray analyses revealed that CI more profoundly altered the pattern of genes expressed by human CECs than CIV. CONCLUSIONS: Collagens considerably suppressed α5 gene expression in CECs, suggesting that during wound healing, they may interfere with the influence FN exerts on CECs by altering their adhesive and migratory properties through a mechanism involving a reduction in α5 gene expression.

Bone Metastasis in Renal Cell Carcinoma is Preprogrammed in the Primary Tumor and Caused by AKT and Integrin α5 Signaling.

PURPOSE: Bone metastasis develops in 30% of all patients with renal cell carcinoma. We elucidated the mechanisms that lead to and predict bone metastasis of renal cell carcinoma. MATERIALS AND METHODS: Nine renal cell carcinoma primary cell lines and 30 renal cell carcinoma tissue specimens (normal and tumor tissue) were collected from 3 patients with no metastasis and 10 with lung or bone metastasis within 5 years after nephrectomy. Cell migration was analyzed in a Boyden chamber and proliferation was assessed by bromodeoxyuridine incorporation. Adhesion to fibronectin, and collagen I and IV was determined after cell staining. The expression and/or activity of cellular signaling molecules was quantified by Western blot. RESULTS: Compared to renal cell carcinoma cells from patients without metastasis, the migration of cells from patients with bone metastasis was enhanced 13.5-fold (p = 0.034), and adhesion to fibronectin and collagen I was enhanced 5.8-fold and 6.1-fold (p = 0.002 and 0.014, respectively). In general proliferation was decreased in metastasizing cells. In accordance with these results we detected higher activity of AKT (p = 0.011) and FAK (p = 0.054), higher integrin α5 expression (p = 0.052) and lower PTEN expression in primary cells from patients with bone metastasis compared to nonmetastasizing cells. An almost similarly altered expression pattern was also observed in the renal cell carcinoma tissue specimens and the normal renal tissue of patients with bone metastasis. CONCLUSIONS: We describe evidence that molecular predispositions determine the potential for bone metastasis to develop in renal cell carcinoma, which may serve as prognostic markers after initial tumor detection.

TM4SF5 suppression disturbs integrin α5-related signalling and muscle development in zebrafish.

TM4SF5 (transmembrane 4 L six family member 5) is involved in EMT (epithelial-mesenchymal transition) for liver fibrosis and cancer metastasis; however, the function(s) of TM4SF5 during embryogenesis remains unknown. In the present study the effects of TM4SF5 on embryogenesis of zebrafish were investigated. tm4sf5 mRNA was expressed in the posterior somites during somitogenesis and in whole myotome 1 dpf (day post-fertilization). tm4sf5 suppression impaired development of the trunk with aberrant morphology of muscle fibres and altered expression of integrin α5. The arrangement and adhesion of muscle cells were abnormally disorganized in tm4sf5 morphants with reduced muscle fibre masses, where integrin α5-related signalling molecules, including fibronectin, FAK (focal adhesion kinase), vinculin and actin were aberrantly localized, compared with those in control fish. Aberrant muscle developments in tm4sf5 morphants were recovered by additional tm4sf5 or integrin α5 mRNA injection. Such a role for TM4SF5 was observed in the differentiation of C2C12 mouse myoblast cells to multinuclear muscle cells. Taken together, the results show that TM4SF5 controls muscle differentiation via co-operation with integrin α5-related signalling.

Cyclic hydrodynamic pressure induced proliferation of bladder smooth muscle cells via integrin alpha5 and FAK.

According to previous studies, integrins play an important role in the mechanotransduction. The aim of this study was to examine the role of integrin subunits and its down-stream signaling molecules in the cyclic hydrodynamic pressure-induced proliferation of human bladder smooth muscle cells (HBSMCs) cultured in scaffolds. The HBSMCs cultured in scaffolds were subjected to four different levels of cyclic hydrodynamic pressure for 24 hours, which were controlled by a BOSE BioDynamic bioreactor. Flow cytometry was used to examine cell cycle distribution. Real-time RT-PCR and western blotting were used to examine the expression levels of integrin subunits and their downstream signaling molecules. Integrin alpha5 siRNA was applied to validate the role of integrin alpha5 in cell proliferation. Here, we showed that cyclic hydrodynamic pressure promoted proliferation of HBSMCs. The cyclic hydrodynamic pressure also increased expression of integrin alpha5 and phosphorylation of FAK, the key mediator of integrin alpha5 signaling, but not that of integrin alpha1, alpha3, alpha4, alphav, beta1 and beta3. Moreover, inhibition of integrin alpha5 decreased the level of p-FAK and abolished proliferation of HBSMCs stimulated by cyclic hydrodynamic pressure. Taken together, we demonstrate for the ?rst time that the integrin alpha5-FAK signaling pathway controls the proliferation of HBSMCs in response to cyclic hydrodynamic pressure.

Oncogenic targeting of BRM drives malignancy through C/EBPß-dependent induction of α5 integrin.

Integrin expression and activity are altered in tumors, and aberrant integrin signaling promotes malignancy. However, how integrins become altered in tumors remains poorly understood. We discovered that oncogenic activation of MEK signaling induces cell growth and survival, and promotes the malignant phenotype of mammary epithelial cells (MECs) by increasing α5 integrin expression. We determined that MEK activates c-Myc to reduce the transcription of the SWI/SNF chromatin remodeling enzyme Brahma (BRM). Our studies revealed that reduced BRM expression and/or activity drives the malignant behavior of MECs by epigenetically promoting C/EBPß expression to directly induce α5 integrin transcription. Consistently, we could show that restoring BRM levels normalized the malignant behavior of transformed MECs in culture and in vivo by preventing C/EBPß-dependent α5 integrin transcription. Our findings identify a novel mechanism whereby oncogenic signaling promotes malignant transformation by regulating transcription of a key chromatin remodeling molecule that regulates integrin-dependent stromal-epithelial interactions.

Significance of expression of ITGA5 and its splice variants in acute myeloid leukemia: a report from the Children's Oncology Group.

Acute myeloid leukemia (AML) encompasses a heterogeneous group of diseases, and novel biomarkers for risk refinement and stratification are needed to optimize patient care. To identify novel risk factors, we performed transcriptome sequencing on 68 diagnostic AML samples and identified 2 transcript variants (-E2 and -E2/3) of the α-subunit (ITGA5) of the very late antigen-5 integrin. We then quantified expression of ITGA5 and these splice variants in specimens from participants of the AAML03P1 trial. We found no association between ITGA5 expression and clinical outcome. In contrast, patients with the highest relative expression (Q4) of the -E2/3 ITGA5 splice variant less likely had low-risk disease than Q1-3 patients (21% vs. 38%, P = 0.027). Q4 patients had worse response to chemotherapy with a higher proportion having persistent minimal residual disease (50% vs. 23%, P = 0.003) and inferior overall survival (at 5 years: 48% vs. 67%, P = 0.015); the latter association was limited to low-risk patients (Q4 vs. Q1-3: 56% vs. 85%, P = 0.043) and was not seen in standard-risk (51% vs. 60%, P = 0.340) or high-risk (33% vs. 38%, P = 0.952) patients. Our exploratory studies indicate that transcriptome sequencing is useful for biomarker discovery, as exemplified by the identification of ITGA5 -E2/3 splice variant as potential novel adverse prognostic marker for low-risk AML that, if confirmed, could serve to further risk-stratify this patient subset.

Calreticulin overexpression correlates with integrin-α5 and transforming growth factor-ß1 expression in the atria of patients with rheumatic valvular disease and atrial fibrillation.

OBJECTIVES: The aim of this study was to determine whether altered calreticulin expression and distribution contribute to the pathogenesis of atrial fibrillation (AF) associated with valvular heart disease (VHD). BACKGROUND: AF affects electrophysiological and structural changes that exacerbate AF. Atrial remodeling reportedly underlies AF generation, but the precise mechanism of atrial remodeling in AF remains unclear. METHODS: Right and left atrial specimens were obtained from 68 patients undergoing valve replacement surgery. The patients were divided into sinus rhythm (SR; n=25), paroxysmal AF (PaAF; n=11), and persistent AF (PeAF; AF lasting >6 months; n=32) groups. Calreticulin, integrin-α5, and transforming growth factor-ß1 (TGF-ß1) mRNA and protein expression were measured. We also performed immunoprecipitation for calreticulin with either calcineurin B or integrin-α5. RESULTS: Calreticulin, integrin-α5, and TGF-ß1 mRNA and protein expression were increased in the AF groups, especially in the left atrium in patients with mitral valve disease. Calreticulin interacted with both calcineurin B and integrin-α5. Integrin-α5 expression correlated with TGF-ß1 expression, while calreticulin expression correlated with integrin-α5 and TGF-ß1 expression. Despite similar cardiac function classifications, calreticulin expression was greater in the PeAF group than in the SR group. CONCLUSIONS: Calreticulin, integrin-α5, and TGF-ß1 expression was increased in atrial tissue in patients with AF and was related to AF type, suggesting that calreticulin is involved in the pathogenesis of AF in VHD patients.

Activation of p53 pathway by Nutlin-3a inhibits the expression of the therapeutic target α5 integrin in colon cancer cells.

Integrins emerge nowadays as crucial actors of tumor aggressiveness and resistance to therapies. Integrin α5ß1, the fibronectin receptor, determines malignant properties of colon carcinoma which is one of the most important causes of cancer-related deaths in the world. Here we show that inhibition of α5 integrin subunit expression by siRNA or α5ß1 integrin function by specific antagonist affects the survival of HCT116 colon cancer cells. We also evidence that pharmacological reactivation of the tumor suppressor p53 by Nutlin-3a inhibits specifically the expression of the α5 integrin subunit both at the transcriptional and protein level. Inversely repression of α5 integrin modulates p53 activity. A clear relationship between p53 activation by Nutlin-3a, α5 repression and cell survival is shown. No such effects are obtained in cells lacking p53 or when another non-genotoxic activator of p53, RITA, is used. Our results emphasize the crucial role of α5ß1 integrin in colon tumors. Data also suggest that interfering with the integrin α5ß1 through the reactivation of p53 by Nutlin-3a may be of valuable interest as a new therapeutic option for colon tumors expressing high level of the integrin and a wild type p53.

Leukemia cell to endothelial cell communication via exosomal miRNAs.

Recent findings indicate that specific microRNAs (miRNAs), such as those of the miR-17-92 cluster, may be responsible for regulating endothelial gene expression during tumor angiogenesis. Secreted miRNAs enclosed in exosomes also have an important role in cell-cell communication. To elucidate whether miRNAs secreted from neoplastic cells transfer into endothelial cells and are functionally active in the recipient cells, we investigated the effect of exosomal miRNAs derived from leukemia cells (K562) on human umbilical vein endothelial cells (HUVECs). As K562 cells released the miR-17-92 cluster, especially miR-92a, into the extracellular environment, K562 cells, transfected with Cy3-labeled pre-miR-92a, were co-cultured with HUVECs. Cy3-miR-92a derived from K562 cells was detected in the cytoplasm of HUVECs, and the Cy3-miR-92a co-localized with the signals of an exosomal marker, CD63. The expression of integrin α5, a target gene for miR-92a, was significantly reduced in HUVECs by exosomal miR-92a, indicating that exogenous miRNA via exosomal transport can function like endogenous miRNA in HUVECs. The most salient feature of this study is the exosome, derived from K562 cells with enforced miR-92a expression, did not affect the growth of HUVECs but did enhance endothelial cell migration and tube formation. Our results support the idea that exosomal miRNAs have an important role in neoplasia-to-endothelial cell communication.

miR148b is a major coordinator of breast cancer progression in a relapse-associated microRNA signature by targeting ITGA5, ROCK1, PIK3CA, NRAS, and CSF1.

Breast cancer is often fatal during its metastatic dissemination. To unravel the role of microRNAs (miRs) during malignancy, we analyzed miR expression in 77 primary breast carcinomas and identified 16 relapse-associated miRs that correlate with survival and/or distinguish tumor subtypes in different datasets. Among them, miR-148b, down-regulated in aggressive breast tumors, was found to be a major coordinator of malignancy. In fact, it is able to oppose various steps of tumor progression when overexpressed in cell lines by influencing invasion, survival to anoikis, extravasation, lung metastasis formation, and chemotherapy response. miR-148b controls malignancy by coordinating a novel pathway involving over 130 genes and, in particular, it directly targets players of the integrin signaling, such as ITGA5, ROCK1, PIK3CA/p110α, and NRAS, as well as CSF1, a growth factor for stroma cells. Our findings reveal the importance of the identified 16 miRs for disease outcome predictions and suggest a critical role for miR-148b in the control of breast cancer progression.

An angiogenic role for the α5ß1 integrin in promoting endothelial cell proliferation during cerebral hypoxia.

Fibronectin is a critical regulator of vascular modelling, both in development and in the adult. In the hypoxic adult central nervous system (CNS), fibronectin is induced on angiogenic vessels, and endothelial cells show strong induction of the two fibronectin receptors α5ß1 and αvß3 integrins. In a previous study, we found that the αvß3 integrin is dispensable for hypoxic-induced cerebral angiogenesis, but a role for the endothelial α5ß1 integrin was suggested. To directly investigate the role of endothelial α5 integrin in cerebral angiogenesis, wild-type mice and mice lacking α5 integrin expression in endothelial cells (α5-EC-KO) were subject to hypoxia (8% O(2)) for 0, 2, 4, 7 or 14 days. Quantification of cerebral vessel density and endothelial-specific proteins claudin-5 and Glut-1 revealed that α5-EC-KO mice displayed an attenuated angiogenic response, which correlated with delayed endothelial proliferation. α5-EC-KO mice showed no defect in the ability to organize a cerebrovascular fibronectin matrix, and no compensatory increase in vascular αvß3 integrin expression. Consistent with these findings, primary α5KO brain endothelial cells (BEC) in culture exhibited delayed growth and proliferation. Taken together, these studies demonstrate an important angiogenic role for the α5ß1 integrin in promoting BEC proliferation in response to cerebral hypoxia.