Interleukin-6 Signaling Pathway Research Articles

Abstract B19: MET and IL6 signaling in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) accounts for 15-20% of breast cancers and is associated with advanced stage at diagnosis and poorer outcome compared to other breast cancer subtypes. There is an unmet need for targeted therapeutic strategies for TNBC patients since current treatment options are restricted to standard chemotherapy. Both receptor tyrosine kinase (RTK) and inflammatory signaling have been shown to promote cancer progression and are promising therapeutic targets. Our laboratory was the first to demonstrate that the MET receptor tyrosine kinase is highly expressed in TNBC. Hepatocyte growth factor (HGF), the MET ligand, is highly expressed in breast carcinoma and breast carcinoma-associated fibroblasts (CAFs) and is able to induce paracrine or autocrine MET signaling. MET/HGF signaling is also connected with the pro-inflammatory cytokine interleukin 6 (IL6). HGF and IL6 have been shown to interact to enhance invasion of lung cancer cells and progression of multiple myeloma. In breast cancer patients, high serum expression of HGF and IL6 distinguishes metastatic breast cancers. Nonetheless, there is a gap in knowledge as to whether MET and IL6 signaling pathways directly or indirectly interact and how MET/IL6 activation promotes TNBC progression. We are examining the novel concept that MET and IL6 signaling pathways act through a positive signaling feedback loop to drive TNBC progression. By understanding the interactions between these signaling networks, we will be able to design therapeutic strategies that target critical signaling nodes in TNBC. Analysis of gene expression profiles in the four molecular TNBC subtypes defined by Burstein et al. revealed that MET, HGF, and IL6 are expressed in each of the TNBC subtypes. Immunohistochemical analysis of HGF and IL6 expression in breast cancer tissues revealed significantly higher HGF and IL6 expression in TNBC compared to ER+ breast cancers. To determine the effect of MET and IL6 inhibition, we established Mammary Architecture and Microenvironment Engineering (MAME) 3D co-culture models of TNBC cells ± fibroblasts. In these models, TNBC cells have high MET expression, moderate to high IL6 expression, and minimal IL6 receptor (IL6R) expression; whereas the CAF cells have high HGF expression and moderate IL6R expression. Our preliminary studies revealed that an IL6 neutralizing antibody (siltuximab) reduced TNBC structure volumes relative to IL6 expression in the TNBC cells, whereas an IL6 receptor (IL6R) neutralizing antibody (tocilizumab) had no effect. These results correlate with IL6 and IL6R expression levels in TNBC cell lines. We evaluated the efficacy of MET inhibition using XL184 (cabozantinib) and observed that XL184 significantly inhibited TNBC growth, proliferation, and invasion of diverse TNBC cell lines, yet was ineffective against MET-negative breast cancer cells. We are currently evaluating the effect of HGF-mediated MET activation on IL6 signaling and inhibition in our 3D TNBC models. To evaluate the effect of MET and/or IL6 inhibition in vivo we utilized a novel xenograft mouse model that expresses human HGF (hHGFtg SCID). In TNBC cell lines MDA-MB-231 and HCC70, IL6R inhibition slowed tumor progression marginally, whereas MET inhibition with XL184 and the combination of XL184 + anti-IL6R drastically inhibited tumor growth. In a model of established tumor growth, we started treatment when tumor reached 500 mm3. Again we observed a significant decrease in tumor growth with XL184 treatment (p<0.005), but also observed a decrease in tumor growth with anti-IL6R treatment (p<0.03). In our current studies we are evaluating the effects of XL184 and siltuximab treatment in TNBC + CAF tumors. These studies will identify the RTK and inflammatory signaling networks that are critical for TNBC progression and are potential targets for combination therapy. Citation Format: Elizabeth A. Tovar, Mansoureh Sameni, Curt J. Essenburg, Anita Chalasani, Erik S. Linklater, David M. Cherba, Aruselvi Anbalagan, Mary E. Winn, Bonnie F. Sloane, Carrie R. Graveel. MET and IL6 signaling in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B19.

Control of influenza infection is impaired by diminished interferon-γ secretion by CD4 T cells in the lungs of toddler mice.

Respiratory viral infections, such as influenza, can lead to delayed viral clearance in toddlers, possibly exacerbating disease morbidity. We hypothesized that defective CD4 T cells in toddlers may contribute to a failure to clear virus at a similar rate to adults. Thus, we developed a young mouse model to examine potential divergent responses between toddlers and adults. We determined that young mice (toddler mice, 21 d old) were actively generating and recruiting effector/memory T cells, whereas memory populations were firmly established in older, adult mice (8-10 wk old). We infected toddler and adult mice with influenza A/PR8/34 (H1N1) and found young mice had elevated morbidity, as measured by enhanced weight loss and lower partial pressure of oxygen levels, throughout the infection, thus, modeling the higher morbidity observed in children (<2 y old) during infection. Early viral loads were comparable to adult mice, but toddler mice failed to clear virus by 10 d postinfection. This delayed clearance corresponded to poor lung recruitment of CD4 T cells, lower antiviral T cell responses, and lower B cell/antibodies in the lungs. Mechanistically, diminished interferon-γ was detected in the lungs of toddler mice throughout the infection and corresponded to intrinsic, rather than extrinsic, CD4 T cell limitations in interferon-γ transcription. Moreover, defects in interferon-γ production appeared downstream from signal transducer and activator of transcription 4 in the interleukin-12 signaling pathway, suggesting maturational delays different from neonates. Importantly, recombinant interferon-γ supplementation rescued CD4 T cell numbers in the lungs and influenza-specific antibody formation. This study highlights the intrinsic limitations in CD4 T cell effector functions that may arise in toddlers and contribute to disease pathology.

Abstract A57: Platinum induces IL-6-signaling mediated activation of ALDH1A1 and enriches the cancer stem cell population in ovarian cancer.

Abstract Purpose: While chemotherapy may succeed initially at decreasing the number of ovarian cancer (OC) cells, it leaves behind tumors enriched in ovarian cancer stem cells (OCSCs), which most likely drive chemoresistance and tumor relapse. An emerging model indicates that non-OCSCs may dedifferentiate and acquire stem cell properties under certain circumstances. Recent studies in OC implicate a critical role for aberrant cytokine interleukin-6 (IL-6) secretion and the IL-6 signaling pathway may play a critical role in converting non-CSCs to CSC. In the current study, we investigated the mechanism contributing to OCSC enrichment after platinum treatment in vivo. We hypothesize that platinum-induced IL-6 secretion activates STAT3 signaling-mediated expression of the cancer stem cell marker ALDH1A1, resulting in CSC enrichment. In addition, based on our previous study in OCSCs (Wang et al., 2014, Cancer Res.), we sought to examine the mechanism by which guadecitabine (SGI-110), a second generation hypomethylating agent HMA, inhibits OCSCs in OC xenograft residuals after platinum treatment. Methods: OC cells (OVCAR4 and A2780) were cultured alone or co-cultured with normal omental fibroblasts (NOFs) or guadecitabine (100nM, 3days)-treated NOFs in the starving condition for 24h and then treated with 3h cisplatin (CDDP; respective IC50 doses of 27.4 and 14.7μM), guadecitabine, IL-6 (100ng/ml), neutralizing antibody (Nab) against IL-6 (IL-6-Nab, 200ng/mL) or IL-6+IL-6-Nab. IL-6 secretion levels were measured by ELISA. FACS was used to analyze OCSC population marked as aldehyde dehydrogenase (ALDH)+ cells, and western blot was used to examine ALDH1A1 and pSTAT3 levels in cells treated as described above. An ALDH1A1 promoter-luciferase reporter (pGL3-ALDH1A1-Luc; -1 to -1031) assay was used to determine whether IL-6 transactivates ALDH1A1 expression in OC cells. OC cell migration under the described conditions was determined by transwell migration assays. Results: We found that CDDP induced (P&amp;lt;0.05) IL-6 secretion by OCs up to 48h post-treatment and up to 96h by NOFs or by co-cultured cells, suggesting a role of the tumor microenvironment in platinum-induced IL-6 secretion. Guadecitabine inhibited (P&amp;lt;0.05) CDDP-induced IL-6 secretion by NOFs alone or in co-culture with OVCAR4, suggesting an inhibitory role of the HMA on IL-6 signaling. By assaying FACS sorted ALDH+/- cells, we determined that ALDH+ cells express increased (P&amp;lt;0.05) levels of the IL-6 receptor and secrete higher (P&amp;lt;0.05) levels of IL-6 in the conditioned media compared to ALDH- cells. Treatment of OC cells with IL-6 enriched the percentage of ALDH+, which was inhibited by IL-6-Nab. Luciferase assay results revealed that IL-6 transactivated (p&amp;lt;0.05) ALDH1A1 reporter gene expression in ALDH- cells, which was blocked by IL-6-Nab. Further, we observed that IL-6- or CDDP-treated OC cells increased (P&amp;lt;0.05) ALDH1A1 and pSTAT3 protein expression, and guadecitabine treatment inhibited (P&amp;lt;0.05) expression of ALDH1A1 and pSTAT3 in OCs. ALDH+ cells showed greater migration potential than ALDH- cells, and guadecitabine inhibited (P&amp;lt;0.05) migration of both ALDH+ and ALDH- cells. Guadecitabine, alone or combination with IL-6-Nab, inhibited (P&amp;lt;0.05) CDDP-induced OVCAR4 migration in co-culture with NOFs. Conclusion: Our data indicate that IL-6 is a potent regulator of ALDH1A1 expression and the OCSC phenotype and further suggest an inhibitory effect of guadecitabine on the conversion of non-OCSCs to OCSCs. The data support a role for IL-6 in OCSC enrichment after platinum treatment and suggest that a combination approach of IL-6 neutralizing antibody with guadecitabine could represent a novel maintenance strategy after chemotherapy for eradicating OCSCs and preventing tumor recurrence. Citation Format: Yinu Wang, Anirban Kumar Mitra, Kenneth P. Nephew. Platinum induces IL-6-signaling mediated activation of ALDH1A1 and enriches the cancer stem cell population in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A57.

Global gene expression analysis of peripheral blood mononuclear cells in rhesus monkey infants with CA16 infection-induced HFMD

Coxsackievirus A16 (CA16) is a dominant pathogen that results in hand, foot, and mouth disease and causes outbreaks worldwide, particularly in the Asia-Pacific region. However, the underlying molecular mechanisms remain unclear. Our previous study has demonstrated that the basic CA16 pathogenic process was successfully mimicked in rhesus monkey infant. The present study focused on the global gene expression changes in peripheral blood mononuclear cells of rhesus monkey infants with hand, foot, and mouth disease induced by CA16 infection at different time points. Genome-wide expression analysis was performed with Agilent whole-genome microarrays and established bioinformatics tools. Nine hundred and forty-eight significant differentially expressed genes that were associated with 5 gene ontology categories, including cell communication, cell cycle, immune system process, regulation of transcription and metabolic process were identified. Subsequently, the mapping of genes related to the immune system process by PANTHER pathway analysis revealed the predominance of inflammation mediated by chemokine and cytokine signaling pathways and the interleukin signaling pathway. Ultimately, co-expressed genes and their networks were analyzed. The results revealed the gene expression profile of the immune system in response to CA16 in rhesus monkey infants and suggested that such an immune response was generated as a result of the positive mobilization of the immune system. This initial microarray study will provide insights into the molecular mechanism of CA16 infection and will facilitate the identification of biomarkers for the evaluation of vaccines against this virus.

Pacritinib, a Dual FLT3/JAK2 Inhibitor, Reduces Irak-1 Signaling in Acute Myeloid Leukemia

Background: A momentous challenge in the treatment of acute myeloid leukemia (AML) is the significant molecular heterogeneity in driving abnormalities and rapid emergence of resistance. Successful clinical translation of novel targeted agents has been impeded by an incomplete understanding of the genetic drivers and the role of the bone marrow microenvironment that modulates response to therapy. There is thus a need for further investigation of the signaling mechanisms contributing to disease pathogenesis. We previously identified growth dependency on interleukin-1 (IL-1) signaling in >50% of the 90 primary AML samples tested. IL-1 signals through IL-1 receptor-associated kinase (IRAK1), a major mediator of innate immunity and inflammatory responses and a potentially critical therapeutic target in hematopoietic malignancies. These results suggest that targeting the commonly dysregulated IL-1 signaling pathway would be therapeutically beneficial to AML patients. However, there is no clinically viable molecule available to selectively inhibit IL-1 signaling. Here we uncovered that pacritinib, a dual FLT3/JAK2 inhibitor, has high specificity and sensitivity to target IRAK1 in various hematopoietic malignancies, including AML. Methods: We used a combination of biochemical, structural biology, and functional approaches to elucidate the mechanism of action of pacritinib and establish its sensitivity and specificity for target inhibition. Kinome screening analysis against 429 recombinant kinases in the presence of 100 nM pacritinib (approximately 50% of the steady state concentration of free pacritinib at the phase 3 dose of 400 mg QD) was followed by titration (1-100 nM) against those kinases that were >50% inhibited at 100 nM. Because pacritinib is an established dual FLT3/JAK2 inhibitor, we then compared the efficacy of pacritinib to the FLT3 inhibitor quizartinib and the JAK2 inhibitor ruxolitinib in 16 AML cell lines and 25 primary AML samples harboring various genetic lesions. The effects of drug treatments were evaluated on cell viability, survival and downstream signaling. Patient samples having IC50below 1000 nM were considered responsive. To validate the selectivity of pacritinib for binding IRAK1, we identified key interacting residues by molecular docking simulation and performed targeted mutagenesis studies. Results: Pacritinib inhibited the activity of FLT3 and all JAK family members except JAK1 at IC50 values of Conclusions: Pacritinib is a specific inhibitor of IRAK1 that blocks IL-1 signaling in AML-derived cell lines and primary patient samples. To our knowledge, pacritinib is the first clinically viable molecule to robustly and selectively inhibit IRAK1. These findings may be extended to a variety of cancer types with a dependence on IRAK1 kinase. Disclosures Druker:Novartis Pharmaceuticals: Research Funding; Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; ARIAD: Research Funding; MolecularMD: Consultancy, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; CTI Biosciences: Consultancy, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; Oncotide Pharmaceuticals: Research Funding; Oregon Health & Science University: Patents & Royalties; Roche TCRC, Inc.: Consultancy, Membership on an entity9s Board of Directors or advisory committees; AstraZeneca: Consultancy; McGraw Hill: Patents & Royalties; Millipore: Patents & Royalties; Sage Bionetworks: Research Funding; Leukemia & Lymphoma Society: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Gilead Sciences: Consultancy, Membership on an entity9s Board of Directors or advisory committees; Fred Hutchinson Cancer Research Center: Research Funding; Bristol-Myers Squibb: Research Funding; Aptose Therapeutics, Inc (formerly Lorus): Consultancy, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees. Singer:CTI BioPharma, Corp: Employment, Equity Ownership. Agarwal:CTI BioPharma: Research Funding.

Monomethylarsonous acid (MMA+3) Inhibits IL-7 Signaling in Mouse Pre-B Cells.

Our previously published data show that As(+3) invivo and invitro, at very low concentrations, inhibits lymphoid, but not myeloid stem cell development in mouse bone marrow. We also showed that the As(+3) metabolite, monomethylarsonous acid (MMA(+3)), was responsible for the observed pre-B cell toxicity caused by As(+3). Interleukin-7 (IL-7) is the primary growth factor responsible for pre-lymphoid development in mouse and human bone marrow, and Signal Transducer and Activator of Transcription 5 (STAT5) is a transcriptional factor in the IL-7 signaling pathway. We found that MMA(+3) inhibited STAT5 phosphorylation at a concentration as low as 50 nM in mouse bone marrow pre-B cells. Inhibition of STAT5 phosphorylation by As(+3) occurred only at a concentration of 500 nM. In the IL-7 dependent mouse pre-B 2E8 cell line, we also found selective inhibition of STAT5 phosphorylation by MMA(+3), and this inhibition was dependent on effects on JAK3 phosphorylation. IL-7 receptor expression on 2E8 cell surface was also suppressed by 50 nM MMA(+3) at 18 h. As further evidence for the inhibition of STAT5, we found that the induction of several genes required in B cell development, cyclin D1, E2A, EBF1, and PAX5, were selectively inhibited by MMA(+3). Since 2E8 cells lack the enzymes responsible for the conversion of As(+3) to MMA(+3) invitro, the results of these studies suggest that As(+3) induced inhibition of pre-B cell formation invivo is likely dependent on the formation of MMA(+3) which in turn inhibits IL-7 signaling at several steps in mouse pre-B cells.

Outside-in? Acute fetal systemic inflammation in very preterm chronically catheterized sheep fetuses is not driven by cells in the fetal blood

The preterm birth syndrome (delivery before 37 weeks gestation) is a major contributor to the global burden of perinatal morbidity and death. The cause of preterm birth is complex, multifactorial, and likely dependent, at least in part, on the gestational age of the fetus. Intrauterine infection is frequent in preterm deliveries that occur at <32 weeks gestation; understanding how the fetus responds to proinflammatory insult will be an important step towards early preterm birth prevention. However, animal studies of infection and inflammation in prematurity commonly use older fetuses that possess comparatively mature immune systems. Aiming to characterize acute fetal responses to microbial agonist at a clinically relevant gestation, we used 92-day-old fetuses (62% of term) to develop a chronically catheterized sheep model of very preterm pregnancy. We hypothesized that any acute fetal systemic inflammatory responses would be driven by signaling from the tissues exposed to Escherichia coli lipopolysaccharide that is introduced into the amniotic fluid. Eighteen ewes that were carrying a single fetus at 92 days of gestation had recovery surgery to place fetal tracheal, jugular, and intraamniotic catheters. Animals were recovered for 24 hours before being administered either intraamniotic E coli lipopolysaccharide (n = 9) or sterile saline solution (n = 9). Samples were collected for 48 hours before euthanasia and necroscopy. Fetal inflammatory responses were characterized by microarray analysis, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay. Intraamniotic lipopolysaccharide reached the distal trachea within 2 hours. Lipopolysaccharide increased tracheal fluid interleukin-8 within 2 hours and generated a robust inflammatory response that was characterized by interleukin-6 signaling pathway activation and up-regulation of cell proliferation but no increases in inflammatory mediator expression in cord blood RNA. In very preterm sheep fetuses, lipopolysaccharide stimulates inflammation in the fetal lung and fetal skin and stimulates a systemic inflammatory response that is not generated by fetal blood cells. These data argue for amniotic fluid-exposed tissues that play a key role in driving acute fetal and intrauterine inflammatory responses.

A network map of Interleukin-10 signaling pathway.

Interleukin-10 (IL-10) is an anti-inflammatory cytokine with important immunoregulatory functions. It is primarily secreted by antigen-presenting cells such as activated T-cells, monocytes, B-cells and macrophages. In biologically functional form, it exists as a homodimer that binds to tetrameric heterodimer IL-10 receptor and induces downstream signaling. IL-10 is associated with survival, proliferation and anti-apoptotic activities of various cancers such as Burkitt lymphoma, non-Hodgkins lymphoma and non-small scell lung cancer. In addition, it plays a central role in survival and persistence of intracellular pathogens such as Leishmania donovani, Mycobacterium tuberculosis and Trypanosoma cruzi inside the host. The signaling mechanisms of IL-10 cytokine are not well explored and a well annotated pathway map has been lacking. To this end, we developed a pathway resource by manually annotating the IL-10 induced signaling molecules derived from literature. The reactions were categorized under molecular associations, activation/inhibition, catalysis, transport and gene regulation. In all, 37 molecules and 76 reactions were annotated. The IL-10 signaling pathway can be freely accessed through NetPath, a resource of signal transduction pathways previously developed by our group.

Whole-genome sequencing reveals oncogenic mutations in mycosis fungoides

AbstractThe pathogenesis of mycosis fungoides (MF), the most common cutaneous T-cell lymphoma (CTCL), is unknown. Although genetic alterations have been identified, none are considered consistently causative in MF. To identify potential drivers of MF, we performed whole-genome sequencing of MF tumors and matched normal skin. Targeted ultra-deep sequencing of MF samples and exome sequencing of CTCL cell lines were also performed. Multiple mutations were identified that affected the same pathways, including epigenetic, cell-fate regulation, and cytokine signaling, in MF tumors and CTCL cell lines. Specifically, interleukin-2 signaling pathway mutations, including activating Janus kinase 3 (JAK3) mutations, were detected. Treatment with a JAK3 inhibitor significantly reduced CTCL cell survival. Additionally, the mutation data identified 2 other potential contributing factors to MF, ultraviolet light, and a polymorphism in the tumor suppressor p53 (TP53). Therefore, genetic alterations in specific pathways in MF were identified that may be viable, effective new targets for treatment.

In silico database screening of potential targets and pathways of compounds contained in plants used for psoriasis vulgaris.

Reviews and meta-analyses of clinical trials identified plants used as traditional medicines (TMs) that show promise for psoriasis. These include Rehmannia glutinosa, Camptotheca acuminata, Indigo naturalis and Salvia miltiorrhiza. Compounds contained in these TMs have shown activities of relevance to psoriasis in experimental models. To further investigate the likely mechanisms of action of the multiple compounds in these TMs, we undertook a computer-based in silico investigation of the proteins known to be regulated by these compounds and their associated biological pathways. The proteins reportedly regulated by compounds in these four TMs were identified using the HIT (Herbal Ingredients' Targets) database. The resultant data were entered into the PANTHER (Protein ANnotation THrough Evolutionary Relationship) database to identify the pathways in which the proteins could be involved. The study identified 237 compounds in the TMs and these retrieved 287 proteins from HIT. These proteins identified 59 pathways in PANTHER with most proteins being located in the Apoptosis, Angiogenesis, Inflammation mediated by chemokine and cytokine, Gonadotropin releasing hormone receptor, and/or Interleukin signaling pathways. All four TMs contained compounds that had regulating effects on Apoptosis regulator BAX, Apoptosis regulator Bcl-2, Caspase-3, Tumor necrosis factor (TNF) or Prostaglandin G/H synthase 2 (COX2). The main proteins and pathways are primarily related to inflammation, proliferation and angiogenesis which are all processes involved in psoriasis. Experimental studies have reported that certain compounds from these TMs can regulate the expression of proteins involved in each of these pathways.

Interleukin-1 Acts via the JNK-2 Signaling Pathway to Induce Aggrecan Degradation by Human Chondrocytes.

Aggrecan enables articular cartilage to bear load and resist compression. Aggrecan loss occurs early in osteoarthritis and rheumatoid arthritis and can be induced by inflammatory cytokines such as interleukin-1 (IL-1). IL-1 induces cleavage of specific aggrecans characteristic of the ADAMTS proteinases. The aim of this study was to identify the intracellular signaling pathways by which IL-1 causes aggrecan degradation by human chondrocytes and to investigate how aggrecanase activity is controlled by chondrocytes. We developed a cell-based assay combining small interfering RNA (siRNA)-induced knockdown with aggrecan degradation assays. Human articular chondrocytes were overlaid with bovine aggrecan after transfection with siRNAs against molecules of the IL-1 signaling pathway. After IL-1 stimulation, released aggrecan fragments were detected with AGEG and ARGS neoepitope antibodies. Aggrecanase activity and tissue inhibitor of metalloproteinases 3 levels were measured by enzyme-linked immunosorbent assay. Low-density lipoprotein receptor-related protein 1 (LRP-1) shedding was analyzed by Western blotting. ADAMTS-5 is a major aggrecanase in human chondrocytes, regulating aggrecan degradation in response to IL-1. The tumor necrosis factor receptor-associated 6 (TRAF-6)/transforming growth factor β-activated kinase 1 (TAK-1)/MKK-4 signaling axis is essential for IL-1-induced aggrecan degradation, while NF-κB is not. Of the 3 MAPKs (ERK, p38, and JNK), only JNK-2 showed a significant role in aggrecan degradation. Chondrocytes constitutively secreted aggrecanase, which was continuously endocytosed by LRP-1, keeping the extracellular level of aggrecanase low. IL-1 induced aggrecanase activity in the medium in a JNK-2-dependent manner, possibly by reducing aggrecanase endocytosis, because IL-1 caused JNK-2-dependent shedding of LRP-1. The signaling axis TRAF-6/TAK-1/MKK-4/JNK-2 mediates IL-1-induced aggrecanolysis. The level of aggrecanase is controlled by its endocytosis, which may be reduced upon IL-1 stimulation because of LRP-1 shedding.

Upregulated interleukin-6 expression contributes to erlotinib resistance in head and neck squamous cell carcinoma

Despite the role of epidermal growth factor receptor (EGFR) signaling in head and neck squamous cell carcinoma (HNSCC) development and progression, clinical trials involving EGFR tyrosine kinase inhibitors (TKIs) have yielded poor results in HNSCC patients. Mechanisms of acquired resistance to the EGFR TKI erlotinib was investigated by developing erlotinib-resistant HNSCC cell lines and comparing their gene expression profiles with their parental erlotinib-sensitive HNSCC cell lines using microarray analyses and subsequent pathway and network analyses. Erlotinib-resistant HNSCC cells displayed a significant upregulation in immune response and inflammatory pathways compared to parental cells. Interleukin-6 (IL-6) was one of thirteen genes that was significantly differentially expressed in all erlotinib-resistant HNSCC cell lines, which was validated using RT-PCR and ELISA. Blockade of IL-6 signaling using the IL-6 receptor antagonist tocilizumab, was able to overcome erlotinib-resistance in erlotinib-resistant SQ20B tumors in vivo. Overall, erlotinib-resistant HNSCC cells display elevated IL-6 expression levels compared to erlotinib-sensitive HNSCC cells and blockade of the IL-6 signaling pathway may be an effective strategy to overcome resistance to erlotinib and possibly other EGFR TKIs for HNSCC therapy.

Ixodes ricinus salivary serpin IRS-2 affects Th17 differentiation via inhibition of the interleukin-6/STAT-3 signaling pathway.

Th17 cells constitute a subset of CD4(+) T lymphocytes that play a crucial role in protection against extracellular bacteria and fungi. They are also associated with tissue injury in autoimmune and inflammatory diseases. Here, we report that serpin from the tick Ixodes ricinus, IRS-2, inhibits Th17 differentiation by impairment of the interleukin-6 (IL-6)/STAT-3 signaling pathway. Following activation, mature dendritic cells produce an array of cytokines, including the pleiotropic cytokine IL-6, which triggers the IL-6 signaling pathway. The major transcription factor activated by IL-6 is STAT-3. We show that IRS-2 selectively inhibits production of IL-6 in dendritic cells stimulated with Borrelia spirochetes, which leads to attenuated STAT-3 phosphorylation and finally to impaired Th17 differentiation. The results presented extend the knowledge about the effect of tick salivary serpins on innate immunity cells and their function in driving adaptive immune responses.

Whole transcriptome analysis reveals an 8-oxoguanine DNA glycosylase-1-driven DNA repair-dependent gene expression linked to essential biological processes

Reactive oxygen species inflict oxidative modifications on various biological molecules, including DNA. One of the most abundant DNA base lesions, 8-oxo-7,8-dihydroguanine (8-oxoG) is repaired by 8-oxoguanine DNA glycosylase-1 (OGG1) during DNA base excision repair (OGG1-BER). 8-OxoG accumulation in DNA has been associated with various pathological and aging processes, although its role is unclear. The lack of OGG1-BER in Ogg1−/− mice resulted in decreased inflammatory responses and increased susceptibility to infections and metabolic disorders. Therefore, we proposed that OGG1 and/or 8-oxoG base may have a role in immune and homeostatic processes. To test our hypothesis, we challenged mouse lungs with OGG1-BER product 8-oxoG base and changes in gene expression were determined by RNA sequencing and data were analyzed by Gene Ontology and statistical tools. RNA-Seq analysis identified 1592 differentially expressed (≥ 3-fold change) transcripts. The upregulated mRNAs were related to biological processes, including homeostatic, immune-system, macrophage activation, regulation of liquid-surface tension, and response to stimulus. These processes were mediated by chemokines, cytokines, gonadotropin-releasing hormone receptor, integrin, and interleukin signaling pathways. Taken together, these findings point to a new paradigm showing that OGG1-BER plays a role in various biological processes that may benefit the host, but when in excess could be implicated in disease and/or aging processes.

Modeling the Dynamics of Acute Phase Protein Expression in Human Hepatoma Cells Stimulated by IL-6

Interleukin-6 (IL-6) is a systemic inflammatory mediator that triggers the human body’s acute phase response to trauma or inflammation. Although mathematical models for IL-6 signaling pathways have previously been developed, reactions that describe the expression of acute phase proteins were not included. To address this deficiency, a recent model of IL-6 signaling was extended to predict the dynamics of acute phase protein expression in IL-6-stimulated HepG2 cells (a human hepatoma cell line). This included reactions that describe the regulation of haptoglobin, fibrinogen, and albumin secretion by nuclear transcription factors STAT3 dimer and C/EBPβ. This new extended model was validated against two different sets of experimental data. Using the validated model, a sensitivity analysis was performed to identify seven potential drug targets to regulate the secretion of haptoglobin, fibrinogen, and albumin. The drug-target binding kinetics for these seven targets was then integrated with the IL-6 kinetic model to rank them based upon the influence of their pairing with drugs on acute phase protein dynamics. It was found that gp80, JAK, and gp130 were the three most promising drug targets and that it was possible to reduce the therapeutic dosage by combining drugs aimed at the top three targets in a cocktail. These findings suggest hypotheses for further experimental investigation.

Correlation of IL-17 Level in Synovia and Severity of Knee Osteoarthritis.

BackgroundThe aim of this study was to evaluate the role of interleukin-17 (IL-17) level in synovia and its relationship with the severity of knee osteoarthritis (OA).Material/MethodsWe enrolled 226 OA patients and 106 controls in this study. The symptomatic/radiation severity of OA was assessed by the Western Ontario McMaster University Osteoarthritis Index (WOMAC) pain score/Kellgren-Lawrence (KL) grading system. Serum IL-17 levels were measured by enzyme-linked immunosorbent assay (ELISA).ResultsSynovia IL-17 levels were significantly higher in OA patients compared with controls (P<0.01), and were negatively correlated with OA severity. IL-17 level gradually decreased among different phases but lacked statistical significance.ConclusionsIL-17 might play a crucial role in the pathogenesis of OA and is closely related to pain. Blocking the IL-17 signaling pathway may delay pain related to OA.

Adipocyte activation of cancer stem cell signaling in breast cancer.

Signaling within the tumor microenvironment has a critical role in cancer initiation and progression. Adipocytes, one of the major components of the breast microenvironment, have been shown to provide pro-tumorigenic signals that promote cancer cell proliferation and invasiveness in vitro and tumorigenicity in vivo. Adipocyte secreted factors such as leptin and interleukin-6 (IL-6) have a paracrine effect on breast cancer cells. In adipocyte-adjacent breast cancer cells, the leptin and IL-6 signaling pathways activate janus kinase 2/signal transducer and activator of transcription 5, promoting the epithelial-mesenchymal transition, and upregulating stemness regulators such as Notch, Wnt and the Sex determining region Y-box 2/octamer binding transcription factor 4/Nanog signaling axis. In this review we will summarize the major signaling pathways that regulate cancer stem cells in breast cancer and describe the effects that adipocyte secreted IL-6 and leptin have on breast cancer stem cell signaling. Finally we will introduce a new potential treatment paradigm of inhibiting the adipocyte-breast cancer cell signaling via targeting the IL-6 or leptin pathways.

Gene expression profile in cardiovascular disease and preeclampsia: A meta-analysis of the transcriptome based on raw data from human studies deposited in Gene Expression Omnibus

IntroductionCardiovascular disease (CVD) and preeclampsia (PE) share common clinical features. We aimed to identify common transcriptomic signatures involved in CVD and PE in humans. MethodsMeta-analysis of individual raw microarray data deposited in GEO, obtained from blood samples of patients with CVD versus controls and placental samples from women with PE versus healthy women with uncomplicated pregnancies. Annotation of cases versus control samples was taken directly from the microarray documentation. Genes that showed a significant differential expression in the majority of experiments were selected for subsequent analysis. Hypergeometric gene list analysis was performed using Bioconductor GOstats package. Bioinformatic analysis was performed in PANTHER. ResultsSeven studies in CVD and 5 studies in PE were eligible for meta-analysis. A total of 181 genes were found to be differentially expressed in microarray studies investigating gene expression in blood samples obtained from patients with CVD compared to controls and 925 genes were differentially expressed between preeclamptic and healthy placentas. Among these differentially expressed genes, 22 were common between CVD and PE. DiscussionBioinformatic analysis of these genes revealed oxidative stress, p-53 pathway feedback, inflammation mediated by chemokines and cytokines, interleukin signaling, B-cell activation, PDGF signaling, Wnt signaling, integrin signaling and Alzheimer disease pathways to be involved in the pathophysiology of both CVD and PE. Metabolism, development, response to stimulus, immune response and cell communication were the associated biologic processes in both conditions. Gene set enrichment analysis showed the following overlapping pathways between CVD and PE: TGF-β-signaling, apoptosis, graft-versus-host disease, allograft rejection, chemokine signaling, steroid hormone synthesis, type I and II diabetes mellitus, VEGF signaling, pathways in cancer, GNRH signaling, Huntingtons disease and Notch signaling. ConclusionCVD and PE share same common traits in their gene expression profile indicating common pathways in their pathophysiology.

Transcriptomic Evaluation of CD34+ Marrow Cells from Myelodysplastic Syndrome (MDS) Patients

Background: Prior studies using microarray platforms have shown alterations of gene expression profiles (GEPs) in MDS CD34+ marrow cells related to clinical outcomes (Sridhar et al, Blood 2009, Pellagatti et al, JCO 2013). Given the increased sensitivity and accuracy of high-throughput RNA sequencing (RNA-Seq) (Mortazavi et al, Nat Meth 2008, Soon et al, Mol Syst Bio 2012) for detecting and quantifying mRNA transcripts, we applied this methodology for evaluating differential gene expression between MDS and normal CD34+ marrow cells.Methods:RNA was isolated from magnetic bead affinity-enriched CD34+ (>90%) marrow aspirate cells (Miltenyi Biotec, Auburn, CA) and amplified using the Smarter Kit (Clontech, Mt View, CA). The amplified product (ds DNA) was fragmented to a size distribution of ~200-300bp using the E220 Focused Ultrasonicator (Covaris Inc, Woburn, MA). End repair, adapter ligation and PCR amplification were performed using the NEBNext Ultra RNA library prep kit for Illumina (New England Biolabs, Ipswich, MA). The indexed cDNA libraries were sequenced (paired end, 100bp) on an Illumina HiSeq2000 platform with median read counts of 69 million. The sequences were aligned to Human Reference sequence hg19 using DNAnexus mapper with gene detection focused on known annotated genes. The differential expression was analyzed using edgeR. DAVID and Ingenuity IPA programs were used for pathway analyses. Gene Set Enrichment Analysis (GSEA) was used to identify biologic processes in our dataset present across phenotypes.Results: Correlations of RNA-Seq data from unamplified to amplified transcripts demonstrated high fidelity of transcripts obtained (Pearson and Spearman R2 = 0.80). After filtering samples for adequate read counts, 12,323 genes were evaluated. Differential expression analysis yielded 719 differentially expressed genes (DEGs) in MDS (n=30) vs normal (n=21) with FDR <.05. Among the DEGs, 548 and 171 were over- and under-expressed ≥2 fold in MDS vs Normal, respectively: 20% of the overexpressed genes were present in >50% of the patients. Hierarchical cluster analysis using these DEGs confirmed clear separation of MDS patients from normals, with 2 differential expression clusters—one region overexpressed and one underexpressed. A distinctive trend toward clustering of the patients was seen which related to their IPSS categories and marrow blast %. In functional pathway analysis of the 2 distinctive gene clusters which distinguished MDS from normal, the underexpressed MDS DEGs demonstrated enrichment of inflammatory cytokines, oxidative stress and interleukin signaling pathways, plus mitochondrial calcium transport; whereas the MDS overexpressed DEG cluster showed enrichment of adherens junction/cytokeletal remodeling, cell cycle control of chromosome replication and DNA damage response pathways. Using GSEA analysis, significantly increased numbers of genes in MDS vs normal, common to those in gene sets present within curated public databases, were involved with TP53 targets and mTOR signaling pathways.Conclusions: Our study demonstrated that RNA-Seq methodology, a high-throughput and more comprehensive technique than most gene expression microarrays, was capable of showing significant and distinctive differences in gene expression between MDS and normal marrow CD34+ cells. Specific clustering of the DEGs was demonstrated to distinguish patient subsets associated with their major clinical features. Further, the stringently identified DEGs shown to be engaged in functional pathways and biologic processes highly relevant for MDS were extant within the patients’ CD34+ cells. These transcriptomic data provide information complementary to exomic mutational findings contributing to improved understanding of biologic mechanisms underlying MDS. DisclosuresNo relevant conflicts of interest to declare.

Interleukin-6 receptor and its ligand interleukin-6 are opposite markers for survival and infiltration with mature myeloid cells in ovarian cancer

An increased level of interleukin-6 (IL-6) in epithelial ovarian cancer (EOC) is correlated with a worse prognosis. IL-6 stimulates tumor-growth and inflammation. We investigated the intricate interaction between the IL-6 signaling pathway and tumor-infiltrating myeloid cells (TIMs) to determine their prognostic impact in EOC. 160 EOC samples were analyzed for the expression of IL-6, its receptor (IL-6R) and downstream signaling via pSTAT3 by immunohistochemistry. Triple color immunofluorescence confocal microscopy was used to identify myeloid cell populations by CD14, CD33, and CD163. The relationship between these markers, tumor-infiltrating immune cells, clinical-pathological characteristics and survival was investigated. EOC displayed a dense infiltration with myeloid cells, in particular of the CD163+ type. The distribution pattern of all myeloid subtypes was comparable among the different histological subtypes. Analysis of the tumor cells revealed a high expression of IL-6R in 15% and of IL-6 in 23% of patients. Interestingly, tumors expressing IL-6 or IL-6R formed two different groups. Tumors with a high expression of IL-6R displayed low mature myeloid cell infiltration and a longer disease-specific survival (DSS), especially in late stage tumors. High expression of IL-6R was an independent prognostic factor for survival by multivariate analyses (hazard ratio = 0.474, p = 0.011). In contrast, tumors with high epithelial IL-6 expression displayed a dense infiltration of mature myeloid cells and were correlated with a shorter DSS. Furthermore, in densely CD8+ T-cell infiltrated tumors, the ratio between these lymphoid cells and CD163+ myeloid cells was predictive for survival. Thus, IL-6 and IL-6R are opposite markers for myeloid cell infiltration and survival.