Regulation of inflammatory signaling by the ST6Gal-I sialyltransferase.

The ST6Gal-I sialyltransferase, an enzyme that adds α2-6-linked sialic acids to N-glycosylated proteins, regulates multiple immunological processes. However, the contribution of receptor sialylation to inflammatory signaling has been under-investigated. In the current study, we uncovered a role for ST6Gal-I in promoting sustained signaling through two prominent inflammatory pathways, NFκB and JAK/STAT. Using the U937 monocytic cell model, we determined that knockdown (KD) of ST6Gal-I expression had no effect on the rapid activation of NFκB by TNF (≤ 30 min), whereas long-term TNF-induced NFκB activation (2-6 hr) was diminished in ST6Gal-I-KD cells. These data align with prior work in epithelial cells showing that α2-6 sialylation of TNFR1 prolongs TNF-dependent NFκB activation. Similar to TNF, long-term, but not short-term, LPS-induced activation of NFκB was suppressed by ST6Gal-I KD. ST6Gal-I KD cells also exhibited reduced long-term IRF3 and STAT3 activation by LPS. Given that ST6Gal-I activity modulated LPS-dependent signaling, we conducted pull-down assays using SNA (a lectin specific for α2-6 sialic acids) to show that the LPS receptor, TLR4, is a substrate for sialylation by ST6Gal-I. We next assessed signaling by IFNγ, IL-6 and GM-CSF, and found that ST6Gal-I-KD had a limited effect on STAT activation induced by these cytokines. To corroborate these findings, signaling was monitored in bone marrow derived macrophages (BMDMs) from mice with myeloid-specific deletion of ST6Gal-I (LysMCre/ST6Gal-Ifl/fl). In agreement with data from U937 cells, BMDMs with ST6Gal-I knockout displayed reduced long-term activation of NFκB by both TNF and LPS, and diminished long-term LPS-dependent STAT3 activation. However, STAT activation induced by IFNγ, IL-6 and GM-CSF was comparable in wild-type and ST6Gal-I knockout BMDMs. These results implicate ST6Gal-I-mediated receptor sialylation in prolonging the activity of select signaling cascades including TNF/NFκB, LPS/NFκB, and LPS/STAT3, providing new insights into ST6Gal-I's role in modulating the inflammatory phenotype of monocytic cells.

Sialylation of EGFR by the ST6Gal-I sialyltransferase promotes EGFR activation and resistance to gefitinib-mediated cell death.

BACKGROUND: The ST6Gal-I sialyltransferase is upregulated in numerous cancers, and high expression of this enzyme correlates with poor patient prognosis in various malignancies, including ovarian cancer. Through its sialylation of a select cohort of cell surface receptors, ST6Gal-I modulates cell signaling to promote tumor cell survival. The goal of the present study was to investigate the influence of ST6Gal-I on another important receptor that controls cancer cell behavior, EGFR. Additionally, the effect of ST6Gal-I on cancer cells treated with the common EGFR inhibitor, gefitinib, was evaluated. RESULTS: Using the OV4 ovarian cancer cell line, which lacks endogenous ST6Gal-I expression, a kinomics assay revealed that cells with forced overexpression of ST6Gal-I exhibited increased global tyrosine kinase activity, a finding confirmed by immunoblotting whole cell lysates with an anti-phosphotyrosine antibody. Interestingly, the kinomics assay suggested that one of the most highly activated tyrosine kinases in ST6Gal-I-overexpressing OV4 cells was EGFR. Based on these findings, additional analyses were performed to investigate the effect of ST6Gal-I on EGFR activation. To this end, we utilized, in addition to OV4 cells, the SKOV3 ovarian cancer cell line, engineered with both ST6Gal-I overexpression and knockdown, as well as the BxPC3 pancreatic cancer cell line with knockdown of ST6Gal-I. In all three cell lines, we determined that EGFR is a substrate of ST6Gal-I, and that the sialylation status of EGFR directly correlates with ST6Gal-I expression. Cells with differential ST6Gal-I expression were subsequently evaluated for EGFR tyrosine phosphorylation. Cells with high ST6Gal-I expression were found to have elevated levels of basal and EGF-induced EGFR activation. Conversely, knockdown of ST6Gal-I greatly attenuated EGFR activation, both basally and post EGF treatment. Finally, to illustrate the functional importance of ST6Gal-I in regulating EGFR-dependent survival, cells were treated with gefitinib, an EGFR inhibitor widely used for cancer therapy. These studies showed that ST6Gal-I promotes resistance to gefitinib-mediated apoptosis, as measured by caspase activity assays. CONCLUSION: Results herein indicate that ST6Gal-I promotes EGFR activation and protects against gefitinib-mediated cell death. Establishing the tumor-associated ST6Gal-I sialyltransferase as a regulator of EGFR provides novel insight into the role of glycosylation in growth factor signaling and chemoresistance.

ST6Gal-I sialyltransferase promotes tumor necrosis factor (TNF)-mediated cancer cell survival via sialylation of the TNF receptor 1 (TNFR1) death receptor.

Activation of the tumor necrosis factor receptor 1 (TNFR1) death receptor by TNF induces either cell survival or cell death. However, the mechanisms mediating these distinct outcomes remain poorly understood. In this study, we report that the ST6Gal-I sialyltransferase, an enzyme up-regulated in numerous cancers, sialylates TNFR1 and thereby protects tumor cells from TNF-induced apoptosis. Using pancreatic and ovarian cancer cells with ST6Gal-I knockdown or overexpression, we determined that α2-6 sialylation of TNFR1 had no effect on early TNF-induced signaling events, including the rapid activation of NF-κB, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and Akt (occurring within 15 min). However, upon extended TNF treatment (6-24 h), cells with high ST6Gal-I levels exhibited resistance to TNF-induced apoptosis, as indicated by morphological evidence of cell death and decreased activation of caspases 8 and 3. Correspondingly, at these later time points, high ST6Gal-I expressers displayed sustained activation of the survival molecules Akt and NF-κB. Additionally, extended TNF treatment resulted in the selective enrichment of clonal variants with high ST6Gal-I expression, further substantiating a role for ST6Gal-I in cell survival. Given that TNFR1 internalization is known to be essential for apoptosis induction, whereas survival signaling is initiated by TNFR1 at the plasma membrane, we examined TNFR1 localization. The α2-6 sialylation of TNFR1 was found to inhibit TNF-induced TNFR1 internalization. Thus, by restraining TNFR1 at the cell surface via sialylation, ST6Gal-I acts as a functional switch to divert signaling toward survival. These collective findings point to a novel glycosylation-dependent mechanism that regulates the cellular response to TNF and may promote cancer cell survival within TNF-rich tumor microenvironments.

The Tumor-Associated Glycosyltransferase ST6Gal-I Regulates Stem Cell Transcription Factors and Confers a Cancer Stem Cell Phenotype.

The glycosyltransferase ST6Gal-I, which adds α2-6-linked sialic acids to substrate glycoproteins, has been implicated in carcinogenesis; however, the nature of its pathogenic role remains poorly understood. Here we show that ST6Gal-I is upregulated in ovarian and pancreatic carcinomas, enriched in metastatic tumors, and associated with reduced patient survival. Notably, ST6Gal-I upregulation in cancer cells conferred hallmark cancer stem-like cell (CSC) characteristics. Modulating ST6Gal-I expression in pancreatic and ovarian cancer cells directly altered CSC spheroid growth, and clonal variants with high ST6Gal-I activity preferentially survived in CSC culture. Primary ovarian cancer cells from patient ascites or solid tumors sorted for α2-6 sialylation grew as spheroids, while cells lacking α2-6 sialylation remained as single cells and lost viability. ST6Gal-I also promoted resistance to gemcitabine and enabled the formation of stably resistant colonies. Gemcitabine treatment of patient-derived xenograft tumors enriched for ST6Gal-I-expressing cells relative to pair-matched untreated tumors. ST6Gal-I also augmented tumor-initiating potential. In limiting dilution assays, subcutaneous tumor formation was inhibited by ST6Gal-I knockdown, whereas in a chemically induced tumor initiation model, mice with conditional ST6Gal-I overexpression exhibited enhanced tumorigenesis. Finally, we found that ST6Gal-I induced expression of the key tumor-promoting transcription factors, Sox9 and Slug. Collectively, this work highlighted a previously unrecognized role for a specific glycosyltransferase in driving a CSC state. Cancer Res; 76(13); 3978-88. ©2016 AACR.

Preferential Recruitment of Neutrophils into the Cerebellum and Brainstem Contributes to the Atypical Experimental Autoimmune Encephalomyelitis Phenotype.

The JAK/STAT pathway is critical for development, regulation, and termination of immune responses, and dysregulation of the JAK/STAT pathway, that is, hyperactivation, has pathological implications in autoimmune and neuroinflammatory diseases. Suppressor of cytokine signaling 3 (SOCS3) regulates STAT3 activation in response to cytokines that play important roles in the pathogenesis of neuroinflammatory diseases, including IL-6 and IL-23. We previously demonstrated that myeloid lineage-specific deletion of SOCS3 resulted in a severe, nonresolving atypical form of experimental autoimmune encephalomyelitis (EAE), characterized by lesions, inflammatory infiltrates, elevated STAT activation, and elevated cytokine and chemokine expression in the cerebellum. Clinically, these mice exhibit ataxia and tremors. In this study, we provide a detailed analysis of this model, demonstrating that the atypical EAE observed in LysMCre-SOCS3(fl/fl) mice is characterized by extensive neutrophil infiltration into the cerebellum and brainstem, increased inducible NO synthase levels in the cerebellum and brainstem, and prominent axonal damage. Importantly, infiltrating SOCS3-deficient neutrophils produce high levels of CXCL2, CCL2, CXCL10, NO, TNF-α, and IL-1ß. Kinetic studies demonstrate that neutrophil infiltration into the cerebellum and brainstem of LysMCre-SOCS3(fl/fl) mice closely correlates with atypical EAE clinical symptoms. Ab-mediated depletion of neutrophils converts the atypical phenotype to the classical EAE phenotype and, in some cases, a mixed atypical/classical phenotype. Blocking CXCR2 signaling ameliorates atypical EAE development by reducing neutrophil infiltration into the cerebellum/brainstem. Thus, neutrophils lacking SOCS3 display elevated STAT3 activation and expression of proinflammatory mediators and play a critical role in the development of atypical EAE.

Therapeutic efficacy of suppressing the Jak/STAT pathway in multiple models of experimental autoimmune encephalomyelitis.

Pathogenic Th cells and myeloid cells are involved in the pathogenesis of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The JAK/STAT pathway is used by numerous cytokines for signaling and is critical for development, regulation, and termination of immune responses. Dysregulation of the JAK/STAT pathway has pathological implications in autoimmune and neuroinflammatory diseases. Many of the cytokines involved in MS/EAE, including IL-6, IL-12, IL-23, IFN-γ, and GM-CSF, use the JAK/STAT pathway to induce biological responses. Thus, targeting JAKs has implications for treating autoimmune inflammation of the brain. We have used AZD1480, a JAK1/2 inhibitor, to investigate the therapeutic potential of inhibiting the JAK/STAT pathway in models of EAE. AZD1480 treatment inhibits disease severity in myelin oligodendrocyte glycoprotein-induced classical and atypical EAE models by preventing entry of immune cells into the brain, suppressing differentiation of Th1 and Th17 cells, deactivating myeloid cells, inhibiting STAT activation in the brain, and reducing expression of proinflammatory cytokines and chemokines. Treatment of SJL/J mice with AZD1480 delays disease onset of PLP-induced relapsing-remitting disease, reduces relapses and diminishes clinical severity. AZD1480 treatment was also effective in reducing ongoing paralysis induced by adoptive transfer of either pathogenic Th1 or Th17 cells. In vivo AZD1480 treatment impairs both the priming and expansion of T cells and attenuates Ag presentation functions of myeloid cells. Inhibition of the JAK/STAT pathway has clinical efficacy in multiple preclinical models of MS, suggesting the feasibility of the JAK/STAT pathway as a target for neuroinflammatory diseases.

AMP-activated protein kinase restricts IFN-γ signaling.

Inflammation in the CNS contributes to neurologic disorders. Neuroinflammation involves the release of inflammatory molecules from glial cells, such as astrocytes and microglia, and can lead to neuronal damage if unabated. In multiple sclerosis, peripheral immune cells, including IFN-γ-producing Th1 cells, infiltrate the CNS and are important in shaping the inflammatory microenvironment, in part through cytokine-mediated interactions with glial cells. Recent evidence suggests that AMP-activated protein kinase (AMPK), a central regulator of energetic metabolism, can regulate inflammatory gene expression. In this study, we identified that IFN-γ induces biphasic AMPK signaling, suggestive of negative-feedback mechanisms. Activation of AMPK suppresses several IFN-γ-induced cytokines and chemokines in primary astrocytes and microglia. IFN-γ regulates gene expression through activation of STAT1, and deletion of AMPK results in a marked increase in basal expression of STAT1. Conversely, activation of AMPK blocks IFN-γ-induced STAT1 expression. Deletion of AMPK leads to increased basal and IFN-γ-induced expression of inflammatory molecules, including TNF-α, CXCL10, and CCL2. AMPK does not affect the phosphorylation of STAT1, but instead attenuates nuclear translocation of STAT1, DNA binding, and subsequent gene expression. In vivo, AMPK signaling during experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, is downregulated in the brain at onset and peak of disease. Diminution of AMPK signaling in vivo correlates with increased expression of IFN-γ and CCL2 in the CNS. Overall, these findings provide the first link between AMPK and STAT1 and may provide important clues about how bioenergetics and inflammation are linked.

SOCS3 deficiency promotes M1 macrophage polarization and inflammation.

Macrophages participate in both the amplification of inflammation at the time of injury and downregulation of the inflammatory response to avoid excess tissue damage. These divergent functions of macrophages are dictated by their microenvironment, especially cytokines, which promote a spectrum of macrophage phenotypes. The M1 proinflammatory phenotype is induced by LPS, IFN-γ, and GM-CSF, and IL-4, IL-13, and M-CSF induce anti-inflammatory M2 macrophages. Suppressors of cytokine signaling (SOCS) proteins function as feedback inhibitors of the JAK/STAT signaling pathway, and they can terminate innate and adaptive immune responses. In this study, we have evaluated the influence of SOCS3 on macrophage polarization and function. Macrophages obtained from LysMCre-SOCS3(fl/fl) mice, which lack SOCS3 in myeloid lineage cells, exhibit enhanced and prolonged activation of the JAK/STAT pathway compared with macrophages from SOCS3(fl/fl) mice. Furthermore, SOCS3-deficient macrophages have higher levels of the M1 genes IL-1ß, IL-6, IL-12, IL-23, and inducible NO synthase owing to enhanced transcriptional activation and chromatin modifications. SOCS3-deficient M1 macrophages also have a stronger capacity to induce Th1 and Th17 cell differentiation than M1 macrophages from SOCS3(fl/fl) mice. Lastly, LPS-induced sepsis is exacerbated in LysMCre-SOCS3(fl/fl) mice and is associated with enhanced STAT1/3 activation and increased plasma levels of M1 cytokines/chemokines such as IL-1ß, TNF-α, IL-6, CCL3, CCL4, and CXCL11. These findings collectively indicate that SOCS3 is involved in repressing the M1 proinflammatory phenotype, thereby deactivating inflammatory responses in macrophages.

Signal transducer and activator of transcription-3/suppressor of cytokine signaling-3 (STAT3/SOCS3) axis in myeloid cells regulates neuroinflammation.

Suppressor of cytokine signaling (SOCS) proteins are feedback inhibitors of the JAK/STAT pathway. SOCS3 has a crucial role in inhibiting STAT3 activation, cytokine signaling, and inflammatory gene expression in macrophages/microglia. To determine the role of SOCS3 in myeloid cells in neuroinflammation, mice with conditional SOCS3 deletion in myeloid cells (LysMCre-SOCS3(fl/fl)) were tested for experimental autoimmune encephalomyelitis (EAE). The myeloid-specific SOCS3-deficient mice are vulnerable to myelin oligodendrocyte glycoprotein (MOG)-induced EAE, with a severe, nonresolving atypical form of disease. In vivo, enhanced infiltration of inflammatory cells and demyelination is prominent in the cerebellum of myeloid-specific SOCS3-deficient mice, as is enhanced STAT3 signaling and expression of inflammatory cytokines/chemokines and an immune response dominated by Th1 and Th17 cells. In vitro, SOCS3-deficient macrophages exhibit heightened STAT3 activation and are polarized toward the classical M1 phenotype. SOCS3-deficient M1 macrophages provide the microenvironment to polarize Th1 and Th17 cells and induce neuronal death. Furthermore, adoptive transfer of M2 macrophages into myeloid SOCS3-deficient mice leads to delayed onset and reduced severity of atypical EAE by decreasing STAT3 activation, Th1/Th17 cells, and proinflammatory mediators in the cerebellum. These findings indicate that myeloid cell SOCS3 provides protection from EAE through deactivation of neuroinflammatory responses.