Alpha Integrin Research Articles

Brief Report: Alternative Splicing of Extra Domain A (EIIIA) of Fibronectin Plays a Tissue-Specific Role in Hematopoietic Homeostasis.

Fibronectin (FN) is a major extracellular matrix protein implicated in cell adhesion and differentiation in the bone marrow (BM) environment. Alternative splicing of FN gene results in the generation of protein variants containing an additional EIIIA domain that sustains cell proliferation or differentiation during physiological or pathological tissue remodeling. To date its expression and role in adult hematopoiesis has not been explored. In our research, we demonstrate that during physiological hematopoiesis a small fraction of BM derived FN contains the EIIIA domain and that mice constitutively including (EIIIA(+/+) ) or excluding (EIIIA(-/-) ) the EIIIA exon present comparable levels of hematopoietic stem cells, myeloid and lymphoid progenitors within BM. Moreover, only minor alterations were detected in blood parameters and in hematopoietic frequencies of BM granulocytes/monocytes and B cells. As opposed to other tissues, unique compensatory mechanisms, such as increased FN accumulation and variable expression of the EIIIA receptors, Toll like receptor-4 and alpha9 integrin subunit, characterized the BM of these mice. Our data demonstrate that FN is a fundamental component of the hematopoietic tissue and that the EIIIA exon may play a key role in modulating hematopiesis in conditions of BM stress or diseases. Stem Cells 2016;34:2263-2268.

Therapeutic Impact of Sphingosine 1-phosphate Receptor Signaling in Multiple Sclerosis.

Multiple sclerosis (MS) is a female predominant autoimmune demyelinating disease of central nervous system. The proper etiology is not clear. The existing therapies with interferon beta (Betaseron, Rebif), glatiramer acetate (copolymer 1, copaxone) are found to be promising for MS patients. The alpha-4 integrin antagonist monoclonal antibody Natalizumab has been found to decrease brain inflammation in relapsing-remitting MS via inhibition of alpha-4 beta- 1 integrinmediated mode of action of antigen -primed T cells to enter into central nervous system through blood brain barrier. The advancement of drug development introduced prospects of CD52 monoclonal antibody Alemtuzumab and CD20 monoclonal antibody Rituximab in MS therapy. The benefit versus risk ratios of these therapeutic monoclonal antibodies are currently under clinical trial. The ongoing researches demonstrated the importance of HMG-CoA reductase inhibitor statins, NF-κBp65 inhibitor NBD peptide, and antagonist of poly-ADP-ribose polymerase (PARP) in experimental autoimmune encephalomyelitis (EAE), animal model for MS. Recently, the clinical trials indicated the therapeutic prospect of G-protein coupled sphingosine 1-phosphate receptor (S1PR) in MS patients. Recent studies showed remyelination through selective activation of oligodendrocyte progenitor cells. In the context, role of S1PR-mediated signals following interaction with natural ligand S1P and agonist Fingolimod (FTY720) gain profound therapeutic importance in prevention of demyelination in MS brain. The S1PR agonist Fingolimod (FTY 720) has recently been approved by Food and Drug Administration for MS therapy. In the review, we provided an insight on S1PR mode of action in the aspect of treatment of autoimmune disorder, re-myelination and regeneration of axons in damaged central nervous system in multiple sclerosis.

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles.

The hair follicle undergoes lifelong cycling and growth. Previous studies have been focused on epithelial stem cells in the hair follicles. Neural crest stem cells (NCSCs) are pluripotent cells that can persist in adult tissues. We have previously demonstrated that human NCSCs can be isolated from hair follicles. Here, we present a protocol to isolate NCSCs from human hair follicles based on their specific surface-marker expression of CD271/HNK1 or CD271/CD49D (alpha4 integrin). NCSCs can be expanded in the culture as neural spheres or attached cells.

HHV-6, Not JC Virus, Causes Demyelination in PML Connections Between HHV-6, HIV-1 and JC Virus in Multiple Sclerosis, Neuro-AIDS and PML A Commentary and Review Offering Guidelines for Treatment

In recent years, two outstandingly effective drugs for the treatment of multiple sclerosis (MS) have appeared on the market: Tysabri® (Biogen Idec/Elan, 2008) and Gilenya® (Novartis, 2010). Tysabri (Natalizumab) is a humanized monoclonal antibody that binds to the cellular adhesion molecule alpha-4 integrin, which is used by lymphocytes to cross vascular walls and penetrate the blood-brain barrier. Tysabri thus reduces the flux of infected or autoimmune lymphocytes into the CNS. Gilenya (Fingolimod) is an immunomodulatory drug that acts on the sphingosine-1-phosphate receptor to sequester lymphocytes in peripheral lymph nodes, thus also indirectly reducing the flux of infected or autoimmune lymphocytes into the CNS. Both drugs have great clinical promise for treating MS, but both have revealed a critical shortcoming: their use increases the risk of Progressive Multifocal Leucoencephalopathy (PML), a demyelinating disease worse than MS and quickly fatal if not controlled. PML arises in individuals whose immune function is compromised, such as patients undergoing chemotherapy for various kinds of cancer. During the early years of the AIDS epidemic (approximately from 1986-1999), the incidence of PML rose in parallel with increasing numbers of immunocompromised AIDS patients, then fell after effective combined aggressive retrovirus treatment (cART) for HIV-1/AIDS became widely available (after 2000-2001). Unfortunately, the incidence of PML is now rising again in conjunction with the increased use of Tysabri and Gilenya in treating MS. It is therefore important to identify effective treatment modalities for PML.

CD4(+)CD25(High) Treg cells in HIV/HTLV co-infected patients with neuropathy: high expression of Alpha4 integrin and lower expression of Foxp3 transcription factor.

BackgroundRegulatory CD4 T cells (Tregs) are critical in maintaining the homeostasis of the immune system. Quantitative or phenotypic alterations and functional impairment of Tregs have been associated with the development of pathologies including those of the central nervous system. Individuals with HIV-1/HTLV-1 co-infection are more prone to develop neurological complications. The aim of this study was to characterize phenotypically Treg cells in HIV-1/HTLV-1 co-infected Mozambican individuals presenting neurological symptoms.MethodsA cross-sectional study was conducted among HIV-infected individuals presentingneurological symptoms, with and without HTLV co-infection, and blood donors. Peripheral bloodmononuclear cells were stained with monoclonal antibodies conjugated with fluorochromes to quantifyTregs and activated T cells by four colors flow cytometry.ResultsHigher Treg cell frequency (10.6 %) was noted in HIV-1/HTLV-1 co-infected group with neurological symptoms when compared to HIV-1 mono-infected group with neurological symptoms (0.38 %, p = 0.003) and control group (0.9 %, p = 0.0105). An inverse correlation between Foxp3 and CD49d expression was observed in all study groups (rh = −0.71, p = 0.001). In addition, increased levels of Treg cells in co-infected patients were strongly associated with total activated CD4 T cells (rh = 0.8, p = 0.01).ConclusionTreg cells in co-infected patients present phenotypic alterations and might have dysfunction marked by low expression of Foxp3 and increased expression of molecules not frequently seen on Treg cells, such as CD49d. These alterations may be related to (1) changes in Treg cell trafficking and migration, possibly making those cells susceptible to HIV infection, and (2) inability to control the activation and proliferation of effector T lymphocytes.

The role of integrins in glioma biology and anti-glioma therapies.

Integrins are a group of molecules expressed by various cells including glioma cells and endothelial cells within the tumor. There are 18 known alpha and beta integrin subunits which form a heterodimer. Integrins regulate different cellular processes such as proliferation, adhesion, motility and survival as shown in numerous preclinical models. Furthermore, integrins control the activity of the transforming growth factor (TGF)-beta pathway and are involved in the process of angiogenesis which is indispensable for continued tumor growth. Because of the high expression levels of some integrins on glioma cells and their numerous functions, inhibition of integrin signaling has been considered a promising strategy for the treatment of glioma patients. Besides blocking antibodies which are currently under clinical investigation in other cancer entities, the integrin inhibitor cilengitide has been tested within several trials in glioblastoma patients over the last years. Cilengitide is a cyclic RDG peptide which targets integrins alphvbeta3 and alphavbeta5. Based on the results of smaller, initial trials suggesting an activity of cilengitide against glioblastoma, 2 larger trials were subsequently performed. However, both trials, which combined temozolomide-based chemoradiation with cilengitide failed to demonstrate an improved outcome with the addition of cilengitide. Ongoing translational analyses suggest that integrin levels in the tumor tissue are neither prognostic nor predict response to cilengitide. While the clinical development of cilengitide has been stopped, integrin inhibition with more effective agents may still be a promising approach in clinical neurooncology.

Glial cell line-derived neurotrophic factor in combination with insulin-like growth factor 1 and basic fibroblast growth factor promote in vitro culture of goat spermatogonial stem cells

Growth factors are increasingly considered as important regulators of spermatogonial stem cells (SSCs). This study investigated the effects of various growth factors (GDNF, IGF1, bFGF, EGF and GFRalpha-1) on purification and colonization of undifferentiated goat SSCs under in vitro and in vivo conditions. Irrespective of the culture condition used, the first signs of developing colonies were observed from day 4 of culture onwards. The number of colonies developed in GDNF + IGF1 + bFGF culture condition was significantly higher than the other groups (p < 0.05). In contrast, the size of colonies developed in GDNF + EGF + LIF culture condition was significantly higher than the other groups (p < 0.05). Immunocytochemical stationing for specific biomarkers of somatic cells (vimentin, alpha-inhibin and α-SMA) and spermatogonial cells (PLZF, THY 1, VASA, alpha-1 integrin, bet-1 integrin and DBA) revealed that both cell types existed in developing colonies, irrespective of the culture condition used. Even though, the relative abundance of VASA, FGFR3, OCT4, PLZF, BCL6B and THY1 transcription factors in GDNF + IGF1 + bFGF treatment group was significantly higher than the other groups (p < 0.05). Additionally, goat SSCs developed in the latter culture condition could colonize within the seminiferous tubules of the germ-cell depleted recipient mice following xenotransplantation. Obtained results demonstrated that combination of GDNF with IGF1 and bFGF promote in vitro culture of goat SSCs while precludes uncontrolled proliferation of somatic cells.

Assessment of Apical Expression of Alpha-2 Integrin, Heat Shock Protein, and Proinflammatory and Immunoregulatory Cytokines in Response to Endodontic Infection

IntroductionThe purpose of this study was to examine alpha-2 integrin, molecular mediators, cytokines, and chemokines from cells in periapical interstitial fluid from root canal infections before and after the reduction of the bacterial load using a cleaning procedure. MethodsSubjects included 20 patients referred to the School of Dentistry at the Universidade Federal de Minas Gerais (Belo Horizonte, Minas Gerais, Brazil). Clinical samples were taken from teeth with pulp necrosis, and no patients had acute periapical symptoms at the time of the appointments. After cleaning and drying, 3 paper points were introduced into the root canal, passing passively through the root apex (2 mm) into the periapical tissues for 1 minute. The samples were collected immediately after root canal cleaning and 7 days later (restrained root canal bacterial load) to characterize those gene expressions using real-time polymerase chain reaction. ResultsSignificantly lower levels of tumor necrosis factor alpha, chemokine ligand 5 (CCL5), chemokine ligand 2/monocyte chemotactic protein 1 (CCL2/MCP-1), and interleukin (IL)-8 in teeth with restrained bacterial loads (second collection) compared with the first collection were observed (P < .05). Similarly, the messenger RNA expression of the integrins secreted phosphoprotein 1 (SSP1)/ostepontin and focal adhesion kinase (FAK) decreased in samples from the second collection (P < .05). The messenger RNA for the regulatory cytokine IL-10 was significant higher in samples from the second collection (day 7) compared with the first collection (day 0) (P < .05). Messenger RNA expression of IL-1β, IL-17A, interferon gamma, alpha-2 integrin, and Hsp47/SERPINH1 were similar at both time points (P > .05). ConclusionsThese findings suggest that after reducing the root canal bacterial load a decrease in the inflammatory response took place in the periapical lesions.

Immunological Markers for PML Prediction in MS Patients Treated with Natalizumab.

Natalizumab (NTZ), a monoclonal antibody recognizing the alpha4 integrin chain, has been approved for the treatment of active multiple sclerosis, but expose to the onset of a rare side effect, progressive multifocal leukoencephalopathy (PML). Estimating the individual risk of PML in NTZ-treated patients is a major challenge, and therapeutic strategies are mainly guided by the overall PML risk assessed by identified risk factors: JC virus (JCV) seropositivity, treatment duration (with peak incidence after 24 months), and the previous use of immunosuppressive therapies. Given that this stratification does not yet allow a precise individual prediction of PML, other predictive markers are needed, and several immunological biomarkers have been described. Quantification of anti-JCV antibody levels may improve individual predictive value, with higher baseline titers indicating increased risk. Other immunological biomarkers such as leukocyte cell membrane markers (CD49d, CD11a, and CD62L), detection of circulating JCV-specific activated T effector memory cells (TEM) or genetic screening have been proposed. In this review, we discuss how recent progress in immunology has paved the way for «new combined monitoring», which will include immunological screening, in NTZ-treated patients.

MicroRNA-26a promotes anoikis in human hepatocellular carcinoma cells by targeting alpha5 integrin.

Metastasis is the major reason for the death of patients suffering from malignant diseases such as human hepatocellular carcinoma (HCC). Among the complex metastatic process, resistance to anoikis is one of the most important steps. Previous studies demonstrate that microRNA-26a (miR-26a) is an important tumor suppressor that inhibits the proliferation and invasion of HCC cells by targeting multiple oncogenic proteins. However, whether miR-26a can also influence anoikis has not been well established. Here, we discovered that miR-26a promotes anoikis of HCC cells both in vitro and in vivo. With a combinational analysis of bioinformatics and public clinical databases, we predicted that alpha5 integrin (ITGA5), an integrin family member, is a putative target of miR-26a. Furthermore, we provide experimental evidence to confirm that ITGA5 is a bona fide target of miR-26a. Through gain- and loss-of-function studies, we demonstrate that ITGA5 is a functional target of miR-26a-induced anoikis in HCC cells. Collectively, our findings reveal that miR-26a is a novel player during anoikis and a potential therapeutic target for the treatment of metastatic HCC.

Bendamustine Improves Clinical Outcome in Chronic Lymphocytic Leukemia (CLL) According to Different Clinical and Biological Prognostic Factors

Bendamustine has been demonstrated to be effective for the treatment of CLL, either alone compared with chlorambucil (Knauf et al, JCO 2009 and BJH 2012) or in combination with monoclonal antibodies such as rituximab both in second or more lines (Fischer et al, JCO 2011) and in first line treatment (Fischer et al, JCO 2012). However, the relationship between its activity with clinical and biological prognosticators has been addressed only in few studies. For this purpose, we evaluated the efficacy and safety of bendamustine, in a real-life contest, on 56 patients, median age 66 years (41-80), median number of previous regimens 1 (0-3, 32% previously untreated). Bendamustine was given for a median number of 6 cycles (70-90 mg/m2), in 82% of cases with rituximab at conventional doses. Overall (ORR) and complete response (CRR) rates were 73% and 44.6%, respectively. Obviously, CRR was higher (83.3%) for 18 patients treated in first line. A significant correlation was found between lower ORR and lymphocyte doubling time <12 months (OR 4.30; P=0.019), thus suggesting that a high proliferation rate may confer a reduced response to bendamustine. As already previously reported, there was a relationship between ORR and number of prior treatments in univariate analysis (OR 0.23; P=0.0055). Interestingly, there was also a significant correlation between lower ORR and the higher expression (>30%) of alpha-4 integrin CD49d (OR 13.0; P=0.018), an important marker of bad prognosis in CLL (Bulian et al, JCO 2014). On the other hand, no significant correlations were found between ORR and CD38, ZAP-70 or IGHV mutational status. Similarly, no significant correlations were noted between ORR and FISH cytogenetics, excluding del(17)p, or NOTCH1 mutations, thus confirming the independence of response to bendamustine from some well-known important biologic prognostic factors. In fact, multivariate analysis confirmed a significant relationship only between ORR and TP53/del(17)p (OR 0.020; P=0.0015) and concomitant rituximab (OR 0.019; P=0.0074). The estimated 1-year OS and PFS were 57% and 86%, respectively. Side effects included grade 3-4 neutropenia, infections, thrombocytopenia and anemia which occurred in 21%, 12%, 12% and 5% of patients, respectively. Grade 3-4 non-hematologic toxicity, including infusion-related reactions, heart or kidney or liver failure were found almost exclusively in elderly patients treated with bendamustine after two or more lines of therapy (12.5%). In multivariate analisys of OS, calculated from the end of treatment with bendamustine, only response to bendamustine (P=0.008) was confirmed to be an independent prognostic factor, while both the number of previous therapies and the concomitant use of rituximab demonstrated no statistical significance. These our results confirm both the activity and safety of bendamustine, particularly in combination with rituximab, also in the setting of elderly patients, often affected by two or three comorbidities. Noteworthy, this effectiveness appears to be present also in patients with unfavorable clinical and biological features, excluding del(17)p or TP53 mutations, in which the employment either of modern oral BCR inhibitors or of BH3 mimetics anti-Bcl-2 will be definitely active, also in combination with the same bendamustine. DisclosuresNo relevant conflicts of interest to declare.

Abstract 17939: Lack of the Alpha-11 Integrin in the Heart is Associated With Progressive Diastolic Dysfunction, Myofibrillar Disarray and Impaired Cardiomyocyte Growth

BACKGROUND: Integrins, transmembrane receptors, play crucial roles in diverse cellular and developmental processes due to critical interactions with the extracellular matrix (ECM). During fetal development and towards adulthood, heart growth and function is suggested to depend on forming and remodeling the ECM and its connection to the myocyte. Currently however, the role of integrins in cardiovascular development (CVD) is poorly defined. Thus, we hypothesized that the α11 integrin (α11), which is expressed by fibroblasts and binds preferentially to type I collagen fibers, plays a vital role in CVD. METHODS: α11 KO and wildtype littermate mice (both n = 8) were examined at 4 weeks and 8 weeks of age. Animals underwent function assessments, including echocardiography and invasive pressure volume (PV) loop analysis, and structural examination via histological and electron microscopy (EM) analysis. RESULTS: At 4 weeks, heart weight (HW) and HW indexed to tibial length were decreased in α11 KO mice (P &lt; 0.05), which were normalized at 8 weeks. Echocardiography revealed reduced end-diastolic area (EDA) at 4 weeks (P &lt; 0.05). Despite normalization of EDA at 8 weeks, PV loop revealed impaired diastolic function as evidence by increased EDP, prolonged Tau and steeper EDPVR (all P &lt; 0.05). No differences in HR or systolic parameters were evident. α11 KO mice also demonstrated structural changes. WGA staining revealed evidence of myofibrillar disarray. Connexin 43 and desmin staining showed increased Z-disk and intermediate filament clustering, respectively. LV myocyte size was also reduced (P &lt; 0.05). Similarly, EM analysis showed reduced cardiomyocyte thickness and distance between end plates (both P &lt; 0.05). CONCLUSION: Loss of α11 resulted in progressively worsening diastolic function that was associated with myofibrillar disarray and impaired cardiomyocyte growth. These findings suggest that α11 is required for the development of normal heart structure and function.

Abstract 215: Aminoflavone exhibits in vivo efficacy on cancer stem cells in a spontaneous estrogen-dependent breast cancer murine model

Abstract Stem-cell populations have been identified in a range of solid tumors and possess the capacity to self renew. It is thought that these cancer stem cells (CSCs) play an important role in cancer establishment, progression, and resistance to current treatments. CSCs have been identified in primary breast cancer tissues and cell lines derived from metastatic tumors. Traditional cancer therapies are effective at debulking some tumors but often fail to produce long-term clinical remissions, presumably due to their inability to eradicate the cancer stem cell population. Therefore, novel treatments aimed at targeting this population can potentially enhance treatment of both primary and metastatic tumors. Aminoflavone (AF; NSC 686288) is a novel anticancer agent. Previous work from our research group demonstrated that AF is a ligand of the aryl hydrocarbon receptor (AhR) and displays cytotoxicity in the MCF-7 breast cancer cell line. A prodrug formulation of AF (AFP464, NSC710464), recently entered phase II clinical trials. Moreover, we previously found that AF disrupts MCF-7 mammosphere formation via AhR signaling activation to inhibit alpha 6-integrin expression. To investigate the efficacy of AF in vivo, we used an estrogen dependent, Tamoxifen-sensitive spontaneous M05 model system involving 1-year-old virgin female BALB/c mice developed in our animal facility. The M05 tumor is a semi-differentiated adenocarcinoma that expresses estrogen and progesterone receptors. After AF treatment (12 mg/kg, i.p., QD × 5 days) tumor size and growth rate decreased substantially compared to control animals. Cytometric analyses of cells obtained from AF-treated and control animals revealed that Lin (-)/ CD29 (Hi)/CD24 (+) cancer stem cells were lower in animals treated with AF than in the vehicle control group. We also observed a diminished capacity for mammosphere formation in cells obtained from tumors treated with AF. These data suggest AF diminishes the quantity of breast cancer stem cells as well as their integrity and capacity to form mammospheres in vitro and in vivo. These findings shed additional insight into the molecular mechanism of anticancer action for this novel anticancer agent to strengthen the rationale for its inclusion in breast cancer treatment regimens. Citation Format: Mariana Callero, Damian Berardi, Laura Todaro, Marina Simian, Eileen J. Brantley, Ubaldo Soto, Andrea Loaiza-Perez. Aminoflavone exhibits in vivo efficacy on cancer stem cells in a spontaneous estrogen-dependent breast cancer murine model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 215. doi:10.1158/1538-7445.AM2014-215

Abstract 37: Bone metastases in renal cell carcinoma are predicted by characteristics of the primary tumor

Abstract Introduction: The poor prognosis of renal cell carcinoma (RCC) is caused by a high risk of metastasis. 30 % of all RCC patients develop bone metastases during course of disease. The outcome of RCC patients with bone metastases is poor so that a prediction would be of advantage. The aim of this study was to elucidate the mechanisms that lead to an organ specific metastasis of RCC in bones and probably predict bone metastasis. Methods: In 30 RCC tissue specimens and 9 RCC primary cell lines collected from patients who had developed no metastases, lung or bone metastases within 5 years after nephrectomy (each 10 RCC tissue specimens, each 3 RCC cell lines) expression and/or activity of 46 cellular signalling molecules were quantified by a phospho kinase array and Western blot analyses. As steps of metastasis, migration of the primary RCC cells was analysed in a Boyden chamber with 10 µg/ml fibronectin as chemotaxin, adhesion to extracellular matrix compounds fibronectin, collagen I and IV was determined after cell staining with crystal violet. Results: In RCC tissue specimens and primary cells from patients who developed bone metastases a higher expression of alpha5 integrins, higher activity of Akt and FAK as well as a lower expression of PTEN was detected, compared to those from patients without or with lung metastases. According to these results we observed an enhanced chemotactic migration (13-fold) of bone metastatic RCC cells and adhesion to fibronectin (6-fold) or collagen type I (8-fold) - both components of the bone matrix. Conclusion: These results show that specific characteristics like the expression and activity of cell signalling mediators and the cellular behaviour of the primary tumor determine the location of the subsequent metastasis in RCC and could be used as prognostic marker for bone metastasis. This should be considered during follow-up care. Funded by the Wilhelm Sander Foundation Citation Format: Elke Schneider, Tobias Haber, Frederick C. Roos, Christian Hampel, Kerstin Junker, Joachim W. Thüroff, Walburgis Brenner. Bone metastases in renal cell carcinoma are predicted by characteristics of the primary tumor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 37. doi:10.1158/1538-7445.AM2014-37

A C-MET-ss1 INTEGRIN COMPLEX DRIVES INVASIVE RESISTANCE TO ANTI-ANGIOGENIC THERAPY

BACKGROUND: Anti-angiogenic therapies like bevacizumab are promising for cancer, but acquired resistance, which often includes increased invasiveness, can limit their utility. We investigated mediators of this invasive resistance using a large archive of bevacizumab-resistant glioblastoma (BRG) tissue and by developing novel xenograft models of anti-angiogenic therapy resistance. METHODS: Microarray analysis, immunostaining, immunoprecipitation, and proximity ligation assays (PLAs) were used to define mediators of invasive anti-angiogenic therapy resistance. RESULTS: Microarray analysis and immunostaining revealed increased hypoxia and reactive-oxygen species (ROS), along with upregulated s1 integrin and receptor tyrosine kinase c-Met, in BRGs compared to pre-treatment (P < 0.005). An siRNA screen and western blot revealed upregulation of AP-1 (P < 0.01), an ROS-activated transcription factor, in BRGs and BRG-derived xenografts with siRNA targeting AP-1 components reducing s1 integrin and c-Met expression in BRG-derived cells. Immunoprecipitation and PLA revealed formation of a c-Met-s1 integrin complex involving s1 heterodimer partners alpha5 and alphaV integrins, with the formation of this complex increased in culture by hypoxia, a feature of anti-angiogenic therapy resistance, and prolonged bevacizumab treatment in vivo. Cross-activation occurred in this complex, with c-Met activation by HGF increasing cell adhesion to fibronectin and fibronectin leading to c-Met phosphorylation. We also found that increasing VEGF concentration suppressed formation of the c-Met-s1 integrin complex (P < 0.05). We developed U87-BevR, a novel xenograft model of acquired bevacizumab resistance through serial multi-generational treatment of U87MG xenografts. U87-BevR exhibited upregulated c-Met and s1 integrin upon development of resistance. Treating U87-BevR with s1 or c-Met inhibitors transiently suppressed invasive growth and reduced c-Met-s1 complex formation. Use of 3- and 10-fold less bevacizumab to treat xenografts led to less initial efficacy but in a more durable fashion associated with less c-Met-s1 integrin complex formation (P < 0.05). CONCLUSIONS: Invasive resistance limits the efficacy of anti-angiogenic therapy. We demonstrated increased activity of s1 integrin and c-Met in BRGs and the formation of a c-Met-s1 complex. The combination of chemotactic c-Met and haptotactic s1 integrin endows BRG cells with their tremendous invasive capacity. The formation of this complex is promoted by two parallel mechanisms–increased transcription of each individual factor driven by hypoxia-induced ROS leading to AP-1 activation and relief of VEGF suppression of the formation of the complex. One option to prevent the formation of a c-Met-s1 complex and thereby counteract the evolution of therapeutic resistance is dose reduction, which may be associated with lesser, but more durable responses, suggesting that, with anti-angiogenic therapy, “less is more.” SECONDARY CATEGORY: Preclinical Experimental Therapeutics.

Mo1214 Long-Term Safety and Efficacy of Golimumab in Patients With Moderately to Severely Active Ulcerative Colitis: Results From the PURSUIT-SC Maintenance Study Extension

Mo1214 Long-Term Safety and Efficacy of Golimumab in Patients With Moderately to Severely Active Ulcerative Colitis: Results From the PURSUIT-SC Maintenance Study Extension

ChemInform Abstract: Functionalized Organozinc Reagents in Medicinal Chemistry: Discovery of Novel Drug Candidates

AbstractReview: [43 refs.

Established and novel disease‐modifying treatments in multiple sclerosis

Multiple sclerosis (MS) is a presumed autoimmune disorder of the central nervous system, resulting in inflammatory demyelination and axonal and neuronal injury. New diagnostic criteria that incorporate magnetic resonance imaging have resulted in earlier and more accurate diagnosis of MS. Several immunomodulatory and immunosuppressive therapeutic agents are available for relapsing forms of MS, which allow individualized treatment based upon the benefits and risks. Disease-modifying therapies introduced in the 1990s, the beta-interferons and glatiramer acetate, have an established track record of efficacy and safety, although they require administration via injection. More recently, monoclonal antibodies have been engineered to act through specific mechanisms such as blocking alpha-4 integrin interactions (natalizumab) or lysing cells bearing specific markers, for example CD52 (alemtuzumab) or CD20 (ocrelizumab and ofatumumab). These agents can be highly efficacious, but sometimes have serious potential complications (natalizumab is associated with progressive multifocal leukoencephalopathy; alemtuzumab is associated with the development of new autoimmune disorders). Three new oral therapies (fingolimod, teriflunomide and dimethyl fumarate, approved for MS treatment from 2010 onwards) provide efficacy, tolerability and convenience; however, as yet, there are no long-term postmarketing efficacy and safety data in a general MS population. Because of this lack of long-term data, in some cases, therapy is currently initiated with the older, safer injectable medications, but patients are monitored closely with the plan to switch therapies if there is any indication of a suboptimal response or intolerance or lack of adherence to the initial therapy. For patients with MS who present with highly inflammatory and potentially aggressive disease, the benefit-to-risk ratio may support initiating therapy using a drug with greater potential efficacy despite greater risks (e.g. fingolimod or natalizumab if JC virus antibody-negative). The aim of this review is to discuss the clinical benefits, mechanisms of action, safety profiles and monitoring strategies of current MS disease-modifying therapies in clinical practice and of those expected to enter the market in the near future.

Par6 is an essential mediator of apoptotic response to transforming growth factor beta in NMuMG immortalized mammary cells

BackgroundWe previously observed that the TGFbeta-Par6 pathway mediates loss of polarity and apoptosis in NMuMG cells. Here we investigate the contribution of Par6 versus TGFbeta receptor I activation to TGFbeta-induced apoptosis in association with changes in apico-basal polarity. We focus on the effect of Par6 activation on alpha6beta4 integrin expression and localization, and Nuclear Factor-kappaB (p65/RelA) activation, previously shown to mediate polarity-dependent cell survival.MethodsUsing immunoblotting and/or immunofluorescence we investigated the effect of TGFbeta1 on apoptosis, alpha6, beta4 and beta1 integrin expression/localization, and p65/RelA phosphorylation/localization in monolayer and three-dimensional (3D) cultures of NMuMG cells with an overactive or inactive Par6 pathway. Results were quantified by band densitometry or as percent of 3D structures displaying a phenotype. Differences among means were compared by two-way ANOVA.ResultsBlocking Par6 activation inhibits TGFbeta-induced apoptosis. Par6 overactivation enhances TGFbeta-induced apoptosis, notably after 6-day exposure to TGFbeta (p < 0.001), a time when parental NMuMG cells no longer respond to TGFbeta apoptotic stimuli. 48-hour TGFbeta treatment reduced beta4 integrin levels in NMuMG monolayers and significantly reduced the basal localization of alpha6 (p < 0.001) and beta4 (p < 0.001) integrin in NMuMG 3D structures, which was dependent on both Par6 and TGFbeta receptor I activation and paralleled apoptotic response. After 6-day exposure to TGFbeta, Par6-dependent changes to beta4 integrin were no longer apparent, but there was reduced phosphorylation of p65/RelA (p < 0.001) only in Par6 overexpressing cells. Differences in p65/RelA localization were not observed among the different cell lines after 48-hour TGFbeta exposure.ConclusionsPar6 and TGFbeta receptor I activation are both necessary for TGFbeta-induced apoptosis in NMuMG cells. Importantly, Par6 overexpression enhances the sensitivity of NMuMG to TGFbeta-induced apoptosis, notably upon prolonged exposure to this growth factor, when NMuMG parental cells are usually apoptosis-resistant. Thus, endogenous Par6 level might be important in determining whether TGFbeta will function as either a pro-apoptotic or pro-survival factor in breast cancer, and potentially aid in predicting patient’s prognosis and therapy response.

Abstract T P237: Alpha-4 Integrin Mediates Inflammatory Monocyte Trafficking to the Brain After Intracerebral Hemorrhage

Background: Intracerebral hemorrhage (ICH) triggers an immune response leading to leukocyte recruitment to the brain and secondary injury. Inflammatory monocytes traffic to the brain over the first 3 days after ICH in murine models, and genetic knock-outs that lack these cells have improved functional outcomes. The aim of this study was to determine the adhesion molecules used by monocytes to enter the brain, and whether antagonism of candidate adhesion molecules could be a new therapeutic target. Methods: ICH was modeled via injection of collagenase into the right striatum of wild type mice. Mice were sacrificed 1 and 3 days post-ICH. Brain and blood were harvested for leukocyte quantification by flow cytometry. Cells were stained with antibodies for CD45, CD3, Ly6G, Ly6C, CD11b, CD49d (alpha-4 integrin), CD11a/CD18 (LFA-1), and CD31 (PECAM). Inflammatory monocytes were identified as CD45high+CD3-Ly6G-CD11b+Ly6C+. Adhesion molecule expression on inflammatory monocytes in brain and blood were compared by t-test. To block leukocyte trafficking, anti-mouse alpha-4 integrin blocking antibody (or IgG control) was administered intraperitoneally 4 hours prior to ICH. Blinded behavioral testing using the cylinder and beam walking tests were used to quantify neurological deficit. Results: Alpha-4 integrin is significantly increased on inflammatory monocytes in the brain after ICH compared to blood on days 1 and 3 (Day 1: 22.7 ±3.4% vs 0.9±0.5%, p&lt;0.001, n=3/group, Day 3: 19.1±4.9% vs 2.9±0.5%, p&lt;0.001, n=5/group). On days 1 and 3 after ICH, the majority of mice could not perform behavioral testing. However, on day 3 mice treated with anti-alpha-4 integrin antibody were more likely to be able to complete 20 rears in the cylinder within 15 minutes compared to controls (50% vs. 0%, p&lt;0.05, n=6/group). Conclusions: Alpha-4 integrin appears to be important for inflammatory monocyte trafficking into the brain after ICH and may be a new potential therapeutic target to prevent the early phase of immune-mediated injury. Importantly, there is a monoclonal antibody therapy approved for use in humans, which could be studied for ICH if further experimental work supports a benefit.