Autocrine Stimulation Research Articles

Abstract PR06: Integrated epigenomic profiling reveals widespread demethylation in melanoma and reveals CSF-1 Receptor as an aberrant regulator of malignant growth and invasion

Abstract Epigenetic changes in cancer are thought to contribute to regulation of tumor invasion and metastasis, but this previously has not been studied at a genome wide level in melanoma. We analyzed the methylome of 44 cases of malignant melanoma with the HELP (HpaII tiny fragment enriched by LM-PCR) assay and compared it with healthy melanocyte controls. We observed widespread demethylation in malignant melanoma, preferentially outside of CpG islands. The epigenomic loss of methylation was independent of mutational status of BRAF, RAS and Kit. Comparison of primary and metastatic lesions demonstrated that demethylation occurs early during carcinogenesis with very few additional alterations in advanced tumors. Parallel transcriptomic analysis revealed many known and novel oncogenic pathways that were aberrantly expressed and regulated by loss of DNA methylation. Strikingly, the colony stimulating factor-1 receptor (CSF1R, c-fms) was aberrantly expressed and hypomethylated in nearly all cases. CSF1R is a transmembrane tyrosine kinase receptor that predominantly regulates macrophages, osteoclasts, and microglia, but is known to sometimes be aberrantly expressed by malignant cells in Hodgkins lymphoma. The expression of CSF1R on malignant melanocytes was validated by immunohistochemical analysis of primary tumors. In several melanoma cell lines (A2058, WM-266-4, SK-MEL-2, M14c#5) we found through PCR sequencing of the cDNA 5′ untranslated region that the CSF1R can be expressed through an aberrant promoter, as has been described for Hodgkin lymphoma. A custom Taqman assay was developed for this unique transcript, and then used to detect the transcript in 4 of 40 samples in a panel of melanoma biopsies, suggesting that aberrant CSF1R expression in melanoma is not uncommon. Expression of CSF1R protein in the cell lines was confirmed by FACS using anti-CD115 antibodies, and by Western blot using antibodies directed to the C-terminus. Expression of the ligand CSF-1 was also found in the melanoma cells by both ELISA and Taqman assays. Inhibition of in vitro cell growth by PLX3397, a clinically relevant small molecule inhibitor of CSF1R kinase, could be observed in 3D cell culture, indicating that under some conditions an autocrine stimulation of growth occurs. shRNA mediated knockdown of CSF1R also demonstrated decreased colony size and increased apoptosis in 3D culture conditions. The invasiveness of melanoma cells was decreased after treatment with PLX3397 or anti-CSF1 antibodies, suggesting a role for melanoma cancer cell expression of CSF1R in metastasis. Since three of cell lines possess an oncogenic BRAF mutation, co-inhibition of CSF1R and BRAF was tested and resulted in synergistic blockade of cell growth in vitro and A2058 xenograft growth in vivo. The CSF1R inhibitor, PLX3397, is under investigation in clinical trials for breast, glioma, and other cancers, and these data present a preclinical rationale for its study in malignant melanoma. This abstract is also being presented as Poster A06. Citation Format: Orsolya Giricz, Yongkai Mo, Caroline Hu, Kimberly Dahlman, Sanchari Bhattacharyya, Hoa Nguyen, Bernice Matusow, Tushar Bhagat, Yiting Yu, Rafe Shellooe, Elizabeth Burton, Gaston Habets, John Greally, Kenny Paraic, Jeffrey Sosman, Gideon Bollag, Brian West, Amit Verma. Integrated epigenomic profiling reveals widespread demethylation in melanoma and reveals CSF-1 Receptor as an aberrant regulator of malignant growth and invasion. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr PR06.

A murine model of acute myeloid leukemia with Evi1 overexpression and autocrine stimulation by an intracellular form of GM-CSF in DA-3 cells

The poor treatment response of acute myeloid leukemia (AML) overexpressing high-risk oncogenes such as EVI1, demands specific animal models for new treatment evaluations. Evi1 is a common site of activating integrations in murine leukemia virus (MLV)-induced AML and in retroviral and lentiviral gene-modified HCS. Still, a model of overt AML induced by Evi1 has not been generated. Cell lines from MLV-induced AML are growth factor-dependent and non-transplantable. Hence, for the leukemia maintenance in the infected animals, a growth factor source such as chronic immune response has been suggested. We have investigated whether these leukemias are transplantable if provided with growth factors. We show that the Evi1+DA-3 cells modified to express an intracellular form of GM-CSF, acquired growth factor independence and transplantability and caused an overt leukemia in syngeneic hosts, without increasing serum GM-CSF levels. We propose this as a general approach for modeling different forms of high-risk human AML using similar cell lines.

Immunological changes accompanying the development of experimental neoplastic process

The key immunology problem remains the understanding of the mechanisms for the effective protection of the body against various pathogens with simultaneous suppression of the immune response to autoantigens. The pathogenesis of neoplastic pathological processes includes violations of the mechanisms of normal cell growth and cell proliferation. Antitumor immune response is a complex event, involving many different cell types. But despite the ability of the immune system to recognize and respond to a variety of tumor-associated antigens, the neoplastic process overcomes the protective forces of the organism, grows and spreads. For cancer cells characterized by independence from antiproliferative signals, autocrine stimulation of growth disturbances in the system, induction of apoptosis and control of genome stability. As a result of accumulation of genetic and epigenetic changes in tumor cells differ significantly from the normal range and the level of expression of genes involved in the transformation process, the accumulation of mutations in key genes promoters and suppressors of tumorigenesis. This creates the opportunity for recognition by cells of the immune system. The study of changes in value and operation of the various elements of the immune system in the development of experimental neoplastic process allows you to identify the mechanisms of interaction in the system «malignant tumor-immune system, to assess patterns of interaction with other organs and tissues, to create a theoretical pathogenetically reasonable premise for the development of anticancer therapy.

Genetic Manipulation of NK Cells for Cancer Immunotherapy: Techniques and Clinical Implications.

Given their rapid and efficient capacity to recognize and kill tumor cells, natural killer (NK) cells represent a unique immune cell to genetically reprogram in an effort to improve the outcome of cell-based cancer immunotherapy. However, technical and biological challenges associated with gene delivery into NK cells have significantly tempered this approach. Recent advances in viral transduction and electroporation have now allowed detailed characterization of genetically modified NK cells and provided a better understanding for how these cells can be utilized in the clinic to optimize their capacity to induce tumor regression in vivo. Improving NK cell persistence in vivo via autocrine IL-2 and IL-15 stimulation, enhancing tumor targeting by silencing inhibitory NK cell receptors such as NKG2A, and redirecting tumor killing via chimeric antigen receptors, all represent approaches that hold promise in preclinical studies. This review focuses on available methods for genetic reprograming of NK cells and the advantages and challenges associated with each method. It also gives an overview of strategies for genetic reprograming of NK cells that have been evaluated to date and an outlook on how these strategies may be best utilized in clinical protocols. With the recent advances in our understanding of the complex biological networks that regulate the ability of NK cells to target and kill tumors in vivo, we foresee genetic engineering as an obligatory pathway required to exploit the full potential of NK-cell based immunotherapy in the clinic.

IFN-β Selectively Inhibits IL-2 Production through CREM-Mediated Chromatin Remodeling.

IFN-β is widely used in the treatment of multiple sclerosis, yet the mechanism facilitating its efficacy remains unclear. IL-2 production by activated T cells, including those mediating autoimmunity, and subsequent autocrine stimulation is vital for T cell expansion and function. In this study, we demonstrate that in mouse and human T cells, IFN-β specifically inhibits the production of IL-2 upon TCR engagement without affecting other cytokines or activation markers. Rather than disrupting TCR signaling, IFN-β alters histone modifications in the IL-2 promoter to retain the locus in an inaccessible configuration. This in turn is mediated through the upregulation of the transcriptional suppressor CREM by IFN-β and consequent recruitment of histone deacetylases to the IL-2 promoter. In accordance, ablation of CREM expression or inhibition of histone deacetylases activity eliminates the suppressive effects of IFN-β on IL-2 production. Collectively, these findings provide a molecular basis by which IFN-β limits T cell responses.

Complement Regulates Nutrient Influx and Metabolic Reprogramming during Th1 Cell Responses

SummaryExpansion and acquisition of Th1 cell effector function requires metabolic reprogramming; however, the signals instructing these adaptations remain poorly defined. Here we found that in activated human T cells, autocrine stimulation of the complement receptor CD46, and specifically its intracellular domain CYT-1, was required for induction of the amino acid (AA) transporter LAT1 and enhanced expression of the glucose transporter GLUT1. Furthermore, CD46 activation simultaneously drove expression of LAMTOR5, which mediated assembly of the AA-sensing Ragulator-Rag-mTORC1 complex and increased glycolysis and oxidative phosphorylation (OXPHOS), required for cytokine production. T cells from CD46-deficient patients, characterized by defective Th1 cell induction, failed to upregulate the molecular components of this metabolic program as well as glycolysis and OXPHOS, but IFN-γ production could be reinstated by retrovirus-mediated CD46-CYT-1 expression. These data establish a critical link between the complement system and immunometabolic adaptations driving human CD4+ T cell effector function.

P0302 : Constitutive gp130 activation accelerates transformation of proliferating human hepatocytes via increased levels of oxidative stress

Background and Aims: Transforming growth factor (TGF)-b is a ubiquitously expressed cytokine with fundamental roles in various aspects of cell physiology. In carcinogenesis, TGF-b signaling plays a dual role. While it suppresses the proliferation of epithelial cells and adenoma cells at early stages by inducing growth arrest and apoptosis, it triggers epithelial to mesenchymal transition (EMT) and gain of metastatic abilities at later stages of carcinoma development. The molecular mechanisms underlying this ’TGF-bswitch’ are only beginning to be unravelled. To mimic the pathophysiological situation as closely as possible, we exposed the cells to TGFb long-term. Through this approach we aimed to identify those cooperating factors and signaling pathways that cause HCC cells to interpret the TGF-b signal in a tumor progressive way. Methods: In vitro comparison of migratory behaviour of various HCC cell lines treated long-term (>10 days) with TGF-b. Analysis of regulatory networks and target genes underlying the TGF-b treatment. Results: HCC cell lines that have undergone EMT secrete TGF-b and show elevated levels of Smad2/3 phosphorylation indicating an autocrine regulatory feedback loop. Inhibition of TGF-b by LY2109 abrogates autocrine stimulation and diminishes the migratory potential of mesenchymal HCC cells. Silencing of either TGF-bR1 or Smad4 indicated the importance of canonical TGF-b/Smad signaling HCC cell migration. Short-term treatment of cells with TGF-b could not improve migratory abilities. Interestingly, long-term TGF-b treatment revealed crucial differences between mesenchymal HCC cell lines. While HLF cells showed an increase in migration when treated with TGF-b for more than 10 days, SNU449 displayed a dramatic reduction in migration. However, both cell lines displayed no modulation in Smad phosphorylation, indicating a change in the utilization of TGF-b signaling in long-term treated SNU449 cells. Conclusions: EMT-transformed HCC cells establish an autocrine TGF-b loop which stimulates migration. However, TGF-b cannot add up to the autocrine loop but causes a different, even opposing reaction, over time. Interpretation of long-term TGF-b signaling, which mimics the patient’s situation more closely, depends on duration and intensity and is controlled by co-acting factors and signaling pathways. P0301 Withdrawn

Autocrine stimulation of IL-10 is critical to the enrichment of IL-10-producing CD40(hi)CD5(+) regulatory B cells in vitro and in vivo.

IL-10-producing B (Breg) cells regulate various immune responses. However, their phenotype remains unclear. CD40 expression was significantly increased in B cells by LPS, and the Breg cells were also enriched in CD40hiCD5+ B cells. Furthermore, CD40 expression on Breg cells was increased by IL-10, CD40 ligand, and B cell-activating factor, suggesting that CD40hi is a common phenotype of Breg cells. LPS-induced CD40 expression was largely suppressed by an anti-IL-10 receptor antibody and in IL-10−/−CD5+CD19+ B cells. The autocrine effect of IL-10 on the CD40 expression was largely suppressed by an inhibitor of JAK/STAT3. In vivo, the LPS treatment increased the population of CD40hiCD5+ Breg cells in mice. However, the population of CD40hiCD5+ B cells was minimal in IL-10−/− mice by LPS. Altogether, our findings show that Breg cells are largely enriched in CD40hiCD5+ B cells and the autocrine effect of IL-10 is critical to the formation of CD40hiCD5+ Breg cells. [BMB Reports 2015; 48(1): 54-59]

Schwann cell Miz without POZ: degeneration meets regeneration.

Peripheral motor and sensory neuropathies are diseases with different etiologies emerging from genetic disorders, diabetes, infection or inflammation, paraneoplastic damage or intoxications including alcohol abuse (Martyn and Hughes, 1997). Hereditary neuropathies, most of which are subsumed under the hypernym Charcot-Marie Tooth (CMT) disease, are clinically heterogeneous: about 50 affected gene loci encoding about 30 genes have been so far identified to be involved in the pathogenesis of different CMTs (Niemann et al., 2006). Depending on structural and electrophysiological features CMTs are divided into axonal and demyelinating forms. Peripheral myelin protein 22 (PMP22), myelin protein zero (MPZ) and connexin 32 (GJB1) are the most frequently affected genes in human demyelinating neuropathies and a variety of animal models have been designed to mimic these human disorders (Bouhy and Timmerman, 2013). These models rely on the deletion, overexpression or mutation of the gene relevant for a human neuropathy and usually clinical symptoms occur early in life and the course of disease is progressive. Recently, Sanz-Moreno et al. (2014) published the partial deletion of the transcription factor Myc interacting zinc finger protein 1 (Miz1) in Schwann cells of mice and observed a late onset demyelinating neuropathy with a spontaneous clinical remission, introducing a unique new model to study peripheral nerve regeneration.

Novel method for real-time monitoring of ATP release reveals multiple phases of autocrine purinergic signalling during immune cell activation.

The activation of immune cells must be tightly regulated to allow an effective immune defence while limiting collateral damage to host tissues. Cellular ATP release and autocrine stimulation of purinergic receptors are recognized as critical regulators of immune cell activation. However, the study of purinergic signalling has been hampered by the short half-life of the released ATP and its breakdown products as well as the lack of real-time imaging methods to study spatiotemporal dynamics of ATP release. To overcome these limitations, we optimized imaging methods that allow monitoring of ATP release with conventional microscopy using the recently developed small molecular ATP probes 1-2Zn(II) and 2-2Zn(II) for imaging of ATP in the extracellular space and release at the surface of living cells. 1-2Zn(II) allowed imaging of <1 μm ATP in the extracellular space, while 2-2Zn(II) provided unprecedented insights into the spatiotemporal dynamics of ATP release from neutrophils and T cells. Stimulation of these cells caused virtually instantaneous ATP release, which was followed by a second phase of ATP release that was localized to the immune synapse of T cells and the leading edge of polarized neutrophils. Imaging these ATP signalling processes along with mitochondrial probes provided evidence for a close spatial relationship between mitochondrial activation and localized ATP release in T cells and neutrophils. We believe that these novel live cell imaging methods can be used to define the roles of purinergic signalling in immune cell activation and in the regulation of other complex physiological processes.

MEDI-573, Alone or in Combination with Mammalian Target of Rapamycin Inhibitors, Targets the Insulin-like Growth Factor Pathway in Sarcomas

MEDI-573 is a human antibody that neutralizes insulin-like growth factor (IGF) I and IGFII. IGFs are overexpressed in multiple types of cancer; their overexpression is a potential mechanism for resistance to IGFI receptor (IGFIR)-targeting therapy. Effects of IGF on cell proliferation, differentiation, and survival are mediated through its binding to and activation of IGFIR or insulin receptor A (IR-A). In this study, we measured the mRNA levels of IGFI, IGFII, and IGFIR in human pediatric sarcoma xenografts, and protein levels in sarcoma cell lines. MEDI-573 potently inhibited in vitro proliferation of sarcoma cell lines, with Ewing sarcoma cell lines being the most sensitive. In addition, MEDI-573 inhibited IGFI- and IGFII-induced sarcoma cell proliferation in vitro. The effect of MEDI-573 on IGF signaling was also examined. Treatment with MEDI-573 markedly reduced levels of pIGFIR, pIR-A, and pAKT and significantly blocked IGFI- and IGFII-induced activation of the IGFIR and AKT pathways. MEDI-573 inhibited the growth of sarcoma xenografts in vivo and inhibition correlated with neutralization of IGFI and IGFII. Combination of MEDI-573 with either rapamycin or AZD2014, another mTOR inhibitor (mTORi), significantly enhanced the antitumor activity of MEDI-573, and this response correlated with modulation of AKT and mTOR signaling. In summary, sarcoma cells respond to autocrine or paracrine growth stimulation by IGFI and IGFII, and inhibition of IGFI and IGFII by MEDI-573 results in significant slowing of tumor growth rate in sarcoma models, particularly in Ewing sarcoma. These data provide evidence for the potential benefits of MEDI-573 and mTORi combinations in patients with Ewing sarcoma.

Purinergic P2Y1 receptors take centre stage in autocrine stimulation of human beta cells.

Insulin secretory vesicles contain high concentrations of adenine nucleotides, which are co-released with insulin during exocytosis. There is strong evidence that ATP and ADP serve as autocrine messengers in pancreatic beta cells, but the functional effects and detailed mechanisms of action are under debate. In this issue of Diabetologia, Khan and colleagues (DOI: 10.1007/s00125-014-3368-8 ) present the results of their study of autocrine purinergic signalling in isolated human beta cells. Using a combination of electrophysiological techniques, Ca(2+) imaging and measurements of insulin secretion, it is demonstrated that voltage-dependent Ca(2+) influx triggers release of ATP/ADP, which activates purinergic receptors of the Gq/11-coupled P2Y1 isoform. Activation of these receptors leads to membrane depolarisation and phospholipase C-mediated mobilisation of Ca(2+) from endoplasmic reticulum stores, which amplifies the exocytosis-triggering Ca(2+) signal. In contrast, there is little evidence for involvement of ionotropic P2X receptors in the autocrine stimulation of human beta cells. This commentary discusses these findings as well as various functional and therapeutic implications of the complex purinergic signalling network in the pancreatic islet.

Temozolomide induces the production of epidermal growth factor to regulate MDR1 expression in glioblastoma cells.

Glioblastoma multiforme (GBM) commonly resists the frontline chemotherapy treatment temozolomide. The multidrug resistance gene (MDR1) and its protein, P-glycoprotein (P-gp), are associated with chemoresistance. This study investigated the mechanisms underlying MDR1-mediated resistance by GBM to temozolomide. P-gp trafficking was studied by flow cytometry and Western blot analysis. MDR1 expression was analyzed by real-time PCR and reporter gene assays. AP-1 interaction with MDR1 was studied by chromatin immunoprecipitation assay. EGF production was analyzed by ELISA, EGFR signaling was determined by Western blot analysis, and in vivo response to erlotinib and/or temozolomide was studied in nude mice. During the early phase of temozolomide treatment, intracellular P-gp was trafficked to the cell membrane, followed by conformational change into active P-gp. At the later phase, gene transcription of MDR1 was induced by temozolomide-mediated production of EGF. EGF activated ERK1/2-JNK-AP-1 cofactors (c-jun and c-fos). An inhibitor of EGFR kinase (erlotinib) given to nude mice with GBM prevented temozolomide-induced resistance. The results identified an essential role for activated EGFR in the resistance of GBM to temozolomide. Temozolomide resistance occurred through a biphasic response; first, by a conformational change in P-gp into the active form and, second, by releasing EGF, which caused autocrine stimulation of GBM cells to induce MDR1. Pharmacologic inhibition of EGFR kinase blunted the ability of GBM cells to resist temozolomide. These findings may explain reports on the common occurrence of mutant EGFR (EGFRvIII) and EGFR expansion in the resistance of GBM cells.

Abstract 4493: Medi-573 alone or in combination with mammalian target of rapamycin inhibitors, targets the insulin-like growth factor pathway in sarcomas

Abstract MEDI-573 is a human antibody that neutralizes the insulin-like growth factors, IGF-1 and IGF-2. IGFs are over-expressed in multiple types of cancer; their over-expression is a potential mechanism for resistance to IGF-1 receptor (IGF-1R)-targeting therapy. Effects of IGFs on cell proliferation, differentiation, and survival are mediated through their binding to and activation of IGF-1R or insulin receptor A (IR-A). In this study, we studied the anti-tumor activity and mechanism of action of MEDI-573 in models of sarcoma. MEDI-573 potently inhibited in vitro proliferation of several sarcoma cell lines, with Ewing's sarcoma cell lines being the most sensitive. This inhibition also occurred after growth stimulation with added IGF-1- and IGF-2. The effect of MEDI-573 on IGF signaling was also examined. Treatment with MEDI-573 markedly reduced levels of pIGF-1R, pIR, and pAKT, and significantly blocked IGF-1- and IGF-2-induced activation of the IGF-1R and AKT pathways. MEDI-573 inhibited the growth of sarcoma xenografts in vivo, and inhibition correlated with neutralization of IGF-1 and IGF-2. Combination of MEDI-573 with either rapamycin or another mTOR inhibitor, AZD2014, significantly enhanced the anti-tumor activity of MEDI-573, and this response correlated with modulation of AKT and mTOR signaling. In summary, sarcoma cells respond to autocrine or paracrine growth stimulation by secreted IGF-1 and IGF-2, and inhibition of IGF-1 and IGF-2 by MEDI-573 results in potent anti-tumor activity in several sarcoma models. Our data provide evidence for evaluation of MEDI-573 and mTORi combinations in clinical studies of sarcoma patients. Note: This abstract was not presented at the meeting. Citation Format: Haihong Zhong, Christine Fazenbaker, Shannon Breen, Cui Chen, Jiaqi Huang, Morehouse Chris, Yihong Yao, Robert Hollingsworth. Medi-573 alone or in combination with mammalian target of rapamycin inhibitors, targets the insulin-like growth factor pathway in sarcomas. [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 4493. doi:10.1158/1538-7445.AM2014-4493

Abstract LB-235: IL10 autoregulatory loop in DLBCLs: New biomarker and a therapeutic target

Abstract Diffuse Large B cell Lymphoma (DLBCL) is a common aggressive lymphoma that represents 30-40% of newly diagnosed cases of non-Hodgkin Lymphomas, but accounts for up to 80% of lymphoma-related mortality. It is biologically and clinically heterogeneous disease with variable response to conventional R-CHOP chemotherapy. R-CHOP remains the standard first line therapy after decades of investigation, but is associated with frequent lack of response. It has been reported that more clinically aggressive cases of DLBCLs have constitutive activation of NF-kB and STAT3 and have higher level of circulating IL10 cytokine in patient's peripheral. We further investigated the role of IL10 and its surface receptor in supporting the neoplastic phenotype of DLBCL. We measured and analyzed copy number changes using SNP array on a subset of 91 primary DLBCLs and identified broad regions of genomic amplification and deletion in this cohort using the GISTIC algorithm and determined that Il10RA is amplified in 17% and IL10RB in 8 % of primary DLBCLs. Gene expression for all 3 genes is markedly elevated, as determined using Affymetrix HG U133 plus 2.0 array data on 59 primary DLBCLs: up to 3 fold for IL10RA, and more than 10 fold for IL10RB and IL10 cytokine as compared to normal Germinal center B cells (NGCB)(all t-test, p&amp;lt;0.01). We also confirmed that IL10R protein expression is elevated using Tissue microarray (TMA) with primary DLBCLs (independent cohort, n=80). Immunohostochemistry on TMA revealed that up to 73% of DLBCLs have overexpression of IL10R several fold above the expression level in normal B cells. ABC DLBCLs tend to have higher level of expression for all 3 genes as compared to GCB DLBCLs. We thus hypothesized that DLBCLs are dependent on the feed-forward autostimulatory loop that starts from autocrine IL10 stimulation through overexpressed receptor leading to cell proliferation and that bliocking the receptor will lead to cell death. We tested the effect of blocking the receptor using anti-IL10R Ab in a panel of 12 cell lines and 5 primary DLBCLs cultured ex-vivo. The Ab effect was dose-dependent and cell death ensued after 1-3 days of treatment. Within 3 days of treatment with 1 ug/ml most cell lines had reduced viability by more than 50%, and after treatment with 10 ug/ml all cell lines were more than 90% dead. On day 3 massive induction of apoptosis was detected in all DLBCLs using standard approaches: measuring Annexin V/ DAPI by flow cytometry and PARP-1 cleavage by western blot. We determined that blocking IL10R results in specific inhibition of signaling through JAK1/2 and loss of phosphorylation at STAT3Y705 immediately after treatment and inhibition of signaling through MAPK and phosphorylation of STAT3S727 at later treatment time points. The inhibition of signaling is sustained for days with only one drug treatment leading to induction of apoptosis. We observed downregulation of the known transcriptional targets of STAT3 that are crucial for maintaining cell cycle and proliferation like CCND1, CCND2, CMYC, JUNB. Anti-IL10R treatment resulted in significant downregulation of IL10 and IL10RA transcription, thus leading to interruption of IL10-IL10R autostimulatory loop. We thus propose that IL10R is a novel therapeutic target in DLBCLs that allows easy detection and targeting. Our findings warranty further animal studies and development of humanized antibody for clinical use in patients. Citation Format: Wendy Beguelin, Seema Sawh, Nyasha Chambwe, Huimin Geng, Yanwen Jiang, Pilar M. Dominguez, Wayne Tam, Rita Shaknovich. IL10 autoregulatory loop in DLBCLs: New biomarker and a therapeutic target. [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 LB-235. doi:10.1158/1538-7445.AM2014-LB-235

Abstract 3726: Inhibition of MET overcomes invasive resistance to Bevacizumab and prolongs survival in orthotopic mouse models of glioblastoma multiforme

Abstract Glioblastoma multiforme (GBM) is the most malignant brain tumor. Despite recent therapeutic improvements, the median overall survival remains poor. Due to its high grade of vascularity, GBM is an ideal candidate for anti-angiogenic therapy. In 2009, US Food and Drug Administration granted accelerated approval for Bevacizumab, a humanized monoclonal antibody directed against VEGF-A, for patients with recurrent disease. Its use in newly diagnosed GBM is currently being evaluated. In the recurrent setting, Bevacizumab provides a transient improvement in PFS that unfortunately does not translate into increased OS. In fact, most tumors become resistant to Bevacizumab and patients invariably experience tumor regrowth. Moreover, a large number of recurrent tumors display an infiltrative and invasive phenotype, thus leading to extremely aggressive tumor growth. Understanding the molecular mechanisms underlying ‘invasive resistance’ to Bevacizumab represents a key step towards the development of improved therapeutic strategies for GBM. We hypothesized that Bevacizumab-induced tumor hypoxia could be responsible for the activation of pro-invasive pathways. We tested this hypothesis using different human GBM cell lines, both in vitro and in orthotopic mouse models of GBM. In vitro, hypoxia promoted increased expression of MET, the high affinity receptor for Hepatocyte Growth Factor(HGF), in all GBM cells analyzed. Increased MET expression resulted in higher sensitivity to HGF stimulation and in enhanced invasiveness. Higher MET levels were not due to transcriptional activation of c-met, but rather to post-transcriptional redistribution of the MET protein from the cytoplasm to the plasma membrane. In vivo, Bevacizumab did marginally improve survival of mice implanted orthotopically with human GBM tumors, but exacerbated tumor hypoxia and resulted in MET activation. Histological analysis of Bevacizumab-treated tumors revealed a highly invasive and infiltrative phenotype. Combined treatment with Bevacizumab and JNJ-38877605, a highly selective, orally available small molecule MET tyrosine kinase inhibitor, significantly prolonged mouse survival compared to both placebo and Bevacizumab alone. Histological examination showed no infiltrative phenotype in the combined treatment arm. Treatment with JNJ-38877605 alone also improved mouse survival in both tumors displaying HGF-MET autocrine stimulation (implanted in nude mice) and in tumors requiring paracrine HGF stimulation (implanted in human HGF knock-in SCID mice). These results unveil a new mechanism by which GBM may become resistant to Bevacizumab, generating proof-of-concept for conducting clinical trials using angiogenesis inhibitors in combination with MET inhibitors. Citation Format: Manuela Cazzanti, Paolo Michieli. Inhibition of MET overcomes invasive resistance to Bevacizumab and prolongs survival in orthotopic mouse models of glioblastoma multiforme. [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 3726. doi:10.1158/1538-7445.AM2014-3726

Shear Stress-Induced NO Production is Dependent on ATP Autocrine Signaling and Capacitative Calcium Entry

Flow-induced production of nitric oxide (NO) by endothelial cells plays a fundamental role in vascular homeostasis. However, the mechanisms by which shear stress activates NO production remain unclear due in part to limitations in measuring NO, especially under flow conditions. Shear stress elicits the release of ATP, but the relative contribution of autocrine stimulation by ATP to flow-induced NO production has not been established. Furthermore, the importance of calcium in shear stress-induced NO production remains controversial, and in particular the role of capacitive calcium entry (CCE) has yet to be determined. We have utilized our unique NO measurement device to investigate the role of ATP autocrine signaling and CCE in shear stress-induced NO production. We found that endogenously released ATP and downstream activation of purinergic receptors and CCE plays a significant role in shear stress-induced NO production. ATP-induced eNOS phophorylation under static conditions is also dependent on CCE. Inhibition of protein kinase C significantly inhibited eNOS phosphorylation and the calcium response. To our knowledge, we are the first to report on the role of CCE in the mechanism of acute shear stress-induced NO response. In addition, our work highlights the importance of ATP autocrine signaling in shear stress-induced NO production.

Sinusoidal Endothelial Cells Coordinate Liver Regeneration and Angiogenesis via Angiopoietin-2: An Ode to Prometheus

Sinusoidal Endothelial Cells Coordinate Liver Regeneration and Angiogenesis via Angiopoietin-2: An Ode to Prometheus

The critical role of macrophage migration inhibitory factor in insulin activity

Macrophage migration inhibitory factor (MIF) is a molecule with plethora of functions such as regulation of immune response, hormone-like, enzymatic and chaperone-like activity. Further, MIF is a major participant in glucose homeostasis since it is an autocrine stimulator of insulin secretion. MIF absence in male knockout mice (MIF-KO) results in development of glucose intolerance, while sensitivity to insulin is fully preserved. Since our results confirm that beta cells from MIF-KO mice express, produce and secrete insulin similarly to beta cells of their wild type (WT) counterparts C57BL/6 mice, we hypothesize that MIF-KO-derived insulin is less active. Indeed, insulin from MIF-KO islets is unable to significantly induce glucose uptake into hepatocytes and to efficiently promote insulin-triggered Akt phosphorylation determined by immunoblot. However, MIF’s tautomerase function is not crucial for insulin biosynthesis since MIF inhibitors had no impact on WT insulin activity. Importantly, MIF recognition by anti-MIF antibody (ELISA) after in vitro co-incubation with purified insulin was significantly lower suggesting that insulin covers MIF immunodominant epitope. In addition, MIF binds insulin within beta cell as confirmed by co-immunoprecipitation. WT and MIF-KO-derived insulin exhibited different cleavage patterns suggesting different protein conformations. Finally, pre-incubation of recombinant MIF with insulin promotes formation of insulin hexamers. These results imply that MIF probably enables proper insulin folding what results in insulin full activity. This newly discovered feature of the cytokine MIF could be potentially important for commercially produced insulin, for increasing its stability and/or bioavailability.

Distinct c-Met activation mechanisms induce cell rounding or invasion through pathways involving integrins, RhoA and HIP1.

Many carcinomas have acquired oncogenic mechanisms for activating c-Met, including c-Met overexpression and excessive autocrine or paracrine stimulation with hepatocyte growth factor (HGF). However, the biological outcome of c-Met activation through these distinct modes remains ambiguous. Here, we report that HGF-mediated c-Met stimulation triggers a mesenchymal-type collective cell invasion. By contrast, the overexpression of c-Met promotes cell rounding. Moreover, in a high-throughput siRNA screen that was performed using a library of siRNAs against putative regulators of integrin activity, we identified RhoA and the clathrin-adapter protein HIP1 as crucial c-Met effectors in these morphological changes. Transient RhoA activation was necessary for the HGF-induced invasion, whereas sustained RhoA activity regulated c-Met-induced cell rounding. In addition, c-Met-induced cell rounding correlated with the phosphorylation of filamin A and the downregulation of active cell-surface integrins. By contrast, a HIP1-mediated increase in β1-integrin turnover was required for the invasion triggered by HGF. Taken together, our results indicate that c-Met induces distinct cell morphology alterations depending on the stimulus that activates c-Met.