TROY (TNFRSF19) is overexpressed in advanced glial tumors and promotes glioblastoma cell invasion via Pyk2-Rac1 signaling

A critical problem in the treatment of malignant gliomas is the extensive infiltration of individual tumor cells into adjacent brain tissues. This invasive phenotype severely limits all current therapies, and to date, no treatment is available to control the spread of this disease. Members of the tumor necrosis factor (TNF) ligand superfamily and their cognate receptors regulate various cellular responses including proliferation, migration, differentiation, and apoptosis. Specifically, the TNFRSF19/TROY gene encodes a type I cell surface receptor that is expressed on migrating or proliferating progenitor cells of the hippocampus, thalamus, and cerebral cortex. Here, we show that levels of TROY mRNA expression directly correlate with increasing glial tumor grade. Among malignant gliomas, TROY expression correlates inversely with overall patient survival. In addition, we show that TROY overexpression in glioma cells activates Rac1 signaling in a Pyk2-dependent manner to drive glioma cell invasion and migration. Pyk2 coimmunoprecipitates with the TROY receptor, and depletion of Pyk2 expression by short hairpin RNA interference oligonucleotides inhibits TROY-induced Rac1 activation and subsequent cellular migration. These findings position aberrant expression and/or signaling by TROY as a contributor, and possibly as a driver, of the malignant dispersion of glioma cells.     

Epidermal Growth Factor (EGF)-enhanced Vascular Cell Adhesion Molecule-1 (VCAM-1) Expression Promotes Macrophage and Glioblastoma Cell Interaction and Tumor Cell Invasion

Activated EGF receptor (EGFR) signaling plays an instrumental role in glioblastoma (GBM) progression. However, how EGFR activation regulates the tumor microenvironment to promote GBM cell invasion remains to be clarified. Here, we demonstrate that the levels of EGFR activation in tumor cells correlated with the levels of macrophage infiltration in human GBM specimens. This was supported by our observation that EGFR activation enhanced the interaction between macrophages and GBM cells. In addition, EGF treatment induced up-regulation of vascular cell adhesion molecule-1 (VCAM-1) expression in a PKCϵ- and NF-κB-dependent manner. Depletion of VCAM-1 interrupted the binding of macrophages to GBM cells and inhibited EGF-induced and macrophage-promoted GBM cell invasion. These results demonstrate an instrumental role for EGF-induced up-regulation of VCAM-1 expression in EGFR activation-promoted macrophage-tumor cell interaction and tumor cell invasion and indicate that VCAM-1 is a potential molecular target for improving cancer therapy.     

TREM-1 Is Induced in Tumor Associated Macrophages by Cyclo-Oxygenase Pathway in Human Non-Small Cell Lung Cancer

It is increasingly recognized that the tumor microenvironment plays a critical role in the initiation and progression of lung cancer. In particular interaction of cancer cells, macrophages, and inflammatory response in the tumor microenvironment has been shown to facilitate cancer cell invasion and metastasis. The specific molecular pathways in macrophages that immunoedit tumor growth are not well defined. Triggering receptor expressed on myeloid cells 1 (TREM-1) is a member of the super immunoglobulin family expressed on a select group of myeloid cells mainly monocyte/macrophages. Recent studies suggest that expression of TREM-1 in tumors may predict cancer aggressiveness and disease outcomes in liver and lung cancer however the mechanism of TREM-1 expression in the setting of cancer is not defined. In this study we demonstrate that tumor tissue from patients with non-small cell lung cancer show an increased expression of TREM-1 and PGE₂. Immunohistochemistry and immunofluorescence confirmed that the expression of TREM-1 was selectively seen in CD68 positive macrophages. By employing an in vitro model we confirmed that expression of TREM-1 is increased in macrophages that are co-cultured with human lung cancer cells. Studies with COX-2 inhibitors and siCOX-2 showed that expression of TREM-1 in macrophages in tumor microenvironment is dependent on COX-2 signaling. These studies for the first time define a link between tumor COX-2 induction, PGE₂ production and expression of TREM-1 in macrophages in tumor microenvironment and suggest that TREM-1 might be a novel target for tumor immunomodulation.     

Astrocytic regulation of human monocytic/microglial activation

Recent reports have described reduced immunological responsiveness and stimulatory capacity among monocytes/microglia that infiltrate malignant human gliomas. Herein, we demonstrate that culture of ex vivo human monocytes or primary human microglia with tumor cells isolated from glioblastoma multiforme (GBM) specimens renders them tolerogenic, capable of suppressing the function of ex vivo monocytes in the absence of tumor cells or their soluble factors. We demonstrate that the tolerance induced in monocytes/microglia by GBM tumor cells is not associated with interference with the signaling cascade associated with TLR- or CD40-induced monocyte activation. Rather, these tumor cells appear to up-regulate pathways that antagonize positive signaling pathways, including but not limited to STAT3 and STAT5. Finally, we demonstrate that the tolerogenic properties of GBM tumor cells amplify properties inherent to nontransformed astrocytes. Future studies that identify all of the molecular mechanisms by which astrocytes and malignant gliomas suppress monocyte/microglial function will have dual therapeutic benefits: suppressing these pathways may benefit patients with astrocytic tumors, while enhancing them may benefit patients with autoimmune processes within the CNS, such as multiple sclerosis.     

NEDD4 promotes cell growth and motility in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related death worldwide. In China, the situation is even worse as cancer incidence and mortality continue to increase rapidly. Although tremendous progress has been made toward HCC treatments, the benefits for liver cancer patients are still limited. Therefore, it is necessary to identify and develop novel therapeutic methods. Neuronally expressed developmentally downregulated 4 (NEDD4), an E3 ubiquitin ligase, plays a critical role in the development and progression of various types of human cancers. In our study, NEDD4 acts as an oncoprotein in both QGY7703 and SMMC7721 liver cancer cell lines. We found that depletion of NEDD4 by siRNA transfection led to inhibition of cell growth, invasion and migration, and promotion of apoptosis. In contrast, overexpression of NEDD4 via plasmid transfection resulted in facilitated cell proliferation, invasion and migration, and decreased apoptosis. Importantly, we observed that tumor suppressor LATS1, also a core component of Hippo pathway, was negatively regulated by NEDD4 in liver cancer cells. Our findings suggested that NEDD4 may be involved in the HCC progression via regulating LATS1 associated signaling pathway. Therefore, targeting NEDD4-LATS1 signaling could be a potential therapeutic option for HCC treatment.     

KCa3.1 channels are involved in the infiltrative behavior of glioblastoma in vivo

Glioblastoma multiforme (GBM) is a diffuse brain tumor characterized by high infiltration in the brain parenchyma rendering the tumor difficult to eradicate by neurosurgery. Efforts to identify molecular targets involved in the invasive behavior of GBM suggested ion channel inhibition as a promising therapeutic approach. To determine if the Ca²⁺-dependent K⁺ channel KCa3.1 could represent a key element for GBM brain infiltration, human GL-15 cells were xenografted into the brain of SCID mice that were then treated with the specific KCa3.1 blocker TRAM-34 (1-((2-chlorophenyl) (diphenyl)methyl)-1H-pyrazole). After 5 weeks of treatment, immunofluorescence analyses of cerebral slices revealed reduced tumor infiltration and astrogliosis surrounding the tumor, compared with untreated mice. Significant reduction of tumor infiltration was also observed in the brain of mice transplanted with KCa3.1-silenced GL-15 cells, indicating a direct effect of TRAM-34 on GBM-expressed KCa3.1 channels. As KCa3.1 channels are also expressed on microglia, we investigated the effects of TRAM-34 on microglia activation in GL-15 transplanted mice and found a reduction of CD68 staining in treated mice. Similar results were observed in vitro where TRAM-34 reduced both phagocytosis and chemotactic activity of primary microglia exposed to GBM-conditioned medium. Taken together, these results indicate that KCa3.1 activity has an important role in GBM invasiveness in vivo and that its inhibition directly affects glioma cell migration and reduces astrocytosis and microglia activation in response to tumor-released factors. KCa3.1 channel inhibition therefore constitutes a potential novel therapeutic approach to reduce GBM spreading into the surrounding tissue.     

PPFIBP1 induces glioma cell migration and invasion through FAK/Src/JNK signaling pathway

Glioblastoma multiforme (GBM) is the most aggressive brain tumor, with a 5-year survival ratio <5%. Invasive growth is a major determinant of the poor prognosis in GBM. In this study, we demonstrate that high expression of PPFIA binding protein 1 (PPFIBP1) correlates with remarkable invasion and poor prognosis of GBM patients. Using scratch and transwell assay, we find that the invasion and migration of GBM cells are promoted by overexpression of PPFIBP1, while inhibited by knockdown of PPFIBP1. Then, we illustrate that overexpression of PPFIBP1 facilitates glioma cell infiltration and reduces survival in xenograft models. Next, RNA-Seq and GO enrichment analysis reveal that PPFIBP1 regulates differentially expressed gene clusters involved in the Wnt and adhesion-related signaling pathways. Furthermore, we demonstrate that PPFIBP1 activates focal adhesion kinase (FAK), Src, c-Jun N-terminal kinase (JNK), and c-Jun, thereby enhancing Matrix metalloproteinase (MMP)-2 expression probably through interacting with SRCIN1 (p140Cap). Finally, inhibition of phosphorylation of Src and FAK significantly reversed the augmentation of invasion and migration caused by PPFIBP1 overexpression in GBM cells. In conclusion, these findings uncover a novel mechanism of glioma invasion and identify PPFIBP1 as a potential therapeutic target of glioma.Subject terms: Oncogenes, Molecular neuroscience     

Increased microglia/macrophage gene expression in a subset of adult and pediatric astrocytomas

Glioblastoma (GBM) is a highly malignant brain tumor with a dismal prognosis. Gene expression profiling of GBM has revealed clinically relevant tumor subtypes, and this provides exciting opportunities to better understand disease pathogenesis. Results from an increasing number of studies demonstrate a role for the immune response in cancer progression, yet it is unclear how the immune response differs across tumor subtypes and how it affects outcome. Utilizing gene expression data from The Cancer Genome Atlas Project and the Gene Expression Omnibus database, we demonstrate an enrichment of immune response-related gene expression in the mesenchymal subtype of adult GBM (n = 173) and pediatric high-grade gliomas (n = 53). In an independent cohort of pediatric astrocytomas (n = 24) from UCSF, we stratified tumors into subtypes and confirmed these findings. Using novel immune cell-specific gene signatures we demonstrate selective enrichment of microglia/macrophage-related genes in adult and pediatric GBM tumors of the mesenchymal subtype. Furthermore, immunostaining of adult GBM tumors showed significantly higher cell numbers of microglia/macrophages in mesenchymal versus non-mesenchymal tumors (p = 0.04). Interestingly, adult GBM tumors with the shortest survival had significant enrichment of microglia/macrophage-related genes but this was not true for pediatric GBMs. Consistent with an association with poor outcome, immune response-related genes were highly represented in an adult poor prognosis gene signature, with the expression of genes related to macrophage recruitment and activation being most strongly associated with survival (p<0.05) using CoxBoost multivariate modeling. Using a microglia/macrophage high gene signature derived from quantification of tumor-infiltrating cells in adult GBM, we identified enrichment of genes characteristic of CD4 T cells, granulocytes, and microglia/macrophages (n = 573). These studies support a role for the immune response, particularly the microglia/macrophage response, in the biology of an important subset of GBM. Identification of this subset may be important for future therapeutic stratification.     

Integrated microenvironment-associated genomic profiles identify LRRC15 mediating recurrent glioblastoma-associated macrophages infiltration

Glioblastoma (GBM) is the most common malignant intracranial tumour with intrinsic infiltrative characteristics, which could lead to most patients eventually relapse. The prognosis of recurrent GBM patients remains unsatisfactory. Cancer cell infiltration and their interaction with the tumour microenvironment (TME) could promote tumour recurrence and treatment resistance. In our study, we aimed to identify potential tumour target correlated with rGBM microenvironment based on the gene expression profiles and clinical information of rGBM patients from The Cancer Genome Atlas (TCGA) database. LRRC15 gene with prognostic value was screened by univariate and multivariate analysis, and the correlation between macrophages and LRRC15 was identified as well. Furthermore, the prognosis correlation and immune characteristics of LRRC15 were validated using the Chinese Glioma Genome Atlas (CGGA) database and our clinical tissues by immunochemistry assay. Additionally, we utilized the transwell assay and carboxy fluorescein succinimidyl ester (CFSE) tracking to further confirm the effects of LRRC15 on attracting microglia/macrophages and tumour cell proliferation in the TME. Gene profiles-based rGBM microenvironment identified that LRRC15 could act in collusion with microglia/macrophages in the rGBM microenvironment to promote the poor prognosis, especially in mesenchymal subtype, indicating the strategies of targeting LRRC15 to improve macrophages-based immunosuppressive effects could be promising for rGBM treatments.     

Fibroblast Growth Factor Receptor 1 Activation in Mammary Tumor Cells Promotes Macrophage Recruitment in a CX3CL1-Dependent Manner

Tumor formation is an extensive process requiring complex interactions that involve both tumor cell-intrinsic pathways and soluble mediators within the microenvironment. Tumor cells exploit the intrinsic functions of many soluble molecules, including chemokines and their receptors, to regulate pro-tumorigenic phenotypes that are required for growth and progression of the primary tumor. Previous studies have shown that activation of inducible FGFR1 (iFGFR1) in mammary epithelial cells resulted in increased proliferation, migration, and invasion in vitro and tumor formation in vivo. These studies also demonstrated that iFGFR1 activation stimulated recruitment of macrophages to the epithelium where macrophages contributed to iFGFR1-mediated epithelial cell proliferation and angiogenesis. The studies presented here further utilize this model to identify the mechanisms that regulate FGFR1-induced macrophage recruitment. Results from this study elucidate a novel role for the inflammatory chemokine CX3CL1 in FGFR1-induced macrophage migration. Specifically, we illustrate that activation of both the inducible FGFR1 construct in mouse mammary epithelial cells and endogenous FGFR in the triple negative breast cancer cell line, HS578T, leads to expression of the chemokine CX3CL1. Furthermore, we demonstrate that FGFR-induced CX3CL1 is sufficient to recruit CX3CR1-expressing macrophages in vitro. Finally, blocking CX3CR1 in vivo leads to decreased iFGFR1-induced macrophage recruitment, which correlates with decreased angiogenesis. While CX3CL1 is a known target of FGF signaling in the wound healing environment, these studies demonstrate that FGFR activation also leads to induction of CX3CL1 in a tumor setting. Furthermore, these results define a novel role for CX3CL1 in promoting macrophage recruitment during mammary tumor formation, suggesting that the CX3CL1/CX3CR1 axis may represent a potential therapeutic approach for targeting breast cancers associated with high levels of tumor-associated macrophages.     

Tumor necrosis factor-alpha stimulates the epithelial-to-mesenchymal transition of human colonic organoids

An epithelial-mesenchymal transition (EMT) characterizes the progression of many carcinomas and it is linked to the acquisition of an invasive phenotype. Given that the tumor microenvironment is an active participant in tumor progression, an important issue is whether a reactive stroma can modulate this process. Using a novel EMT model of colon carcinoma spheroids, we demonstrate that their transforming-growth factor-beta1 (TGF-beta)-induced EMT is accelerated dramatically by the presence of activated macrophages, and we identify tumor necrosis factor-alpha (TNF-alpha) as the critical factor produced by macrophages that accelerates the EMT. A synergy of TNF-alpha and TGF-beta signaling promotes a rapid morphological conversion of the highly organized colonic epithelium to dispersed cells with a mesenchymal phenotype, and this process is dependent on enhanced p38 MAPK activity. Moreover, exposure to TNF-alpha stimulates a rapid burst of ERK activation that results in the autocrine production of this cytokine by the tumor cells themselves. These results establish a novel role for the stroma in influencing EMT in colon carcinoma, and they identify a selective advantage to the stromal presence of infiltrating leukocytes in regulating malignant tumor progression.     

USP18 lack in microglia causes destructive interferonopathy of the mouse brain

Microglia are tissue macrophages of the central nervous system (CNS) that control tissue homeostasis. Microglia dysregulation is thought to be causal for a group of neuropsychiatric, neurodegenerative and neuroinflammatory diseases, called “microgliopathies”. However, how the intracellular stimulation machinery in microglia is controlled is poorly understood. Here, we identified the ubiquitin‐specific protease (Usp) 18 in white matter microglia that essentially contributes to microglial quiescence. We further found that microglial Usp18 negatively regulates the activation of Stat1 and concomitant induction of interferon‐induced genes, thereby terminating IFN signaling. The Usp18‐mediated control was independent from its catalytic activity but instead required the interaction with Ifnar2. Additionally, the absence of Ifnar1 restored microglial activation, indicating a tonic IFN signal which needs to be negatively controlled by Usp18 under non‐diseased conditions. These results identify Usp18 as a critical negative regulator of microglia activation and demonstrate a protective role of Usp18 for microglia function by regulating the Ifnar pathway. The findings establish Usp18 as a new molecule preventing destructive microgliopathy.     

A critical role of Gbetagamma in tumorigenesis and metastasis of breast cancer

A growing body of evidence indicates that G protein-coupled receptors (GPCRs) are involved in breast tumor progression and that targeting GPCRs may be a novel adjuvant strategy in cancer treatment. However, due to the redundant role of multiple GPCRs in tumor development, it may be necessary to target a common signaling component downstream of these receptors to achieve maximum efficacy. GPCRs transmit signals through heterotrimeric G proteins composed of Gα and Gβγ subunits. Here we evaluated the role of Gβγ in breast tumor growth and metastasis both in vitro and in vivo. Our data show that blocking Gβγ signaling with Gα(t) or small molecule inhibitors blocked serum-induced breast tumor cell proliferation as well as tumor cell migration induced by various GPCRs in vitro. Moreover, induced expression of Gα(t) in MDA-MB-231 cells inhibited primary tumor formation and retarded growth of existing breast tumors in nude mice. Blocking Gβγ signaling also dramatically reduced the incidence of spontaneous lung metastasis from primary tumors and decreased tumor formation in the experimental lung metastasis model. Additional studies indicate that Gβγ signaling may also play a role in the generation of a tumor microenvironment permissive for tumor progression, because the inhibition of Gβγ signaling attenuated leukocyte infiltration and angiogenesis in primary breast tumors. Taken together, our data demonstrate a critical role of Gβγ signaling in promoting breast tumor growth and metastasis and suggest that targeting Gβγ may represent a novel therapeutic approach for breast cancer.     

Glutamate production by HIV-1 infected human macrophage is blocked by the inhibition of glutaminase

Mononuclear phagocyte (macrophages and microglia) dysfunction plays a significant role in the pathogenesis of human immunodeficiency virus (HIV) associated dementia (HAD) through the production and release of soluble neurotoxic factors including glutamate. The mechanism of glutamate regulation by HIV-1 infection remains unclear. In this report, we investigated whether the enzyme glutaminase is responsible for glutamate generation by HIV-1 infected monocyte-derived macrophages. We tested the functionality of novel small molecule inhibitors designed to specifically block the activity of glutaminase. Glutaminase inhibitors were first characterized in a kinetic assay with crude glutaminase from rat brain revealing an uncompetitive mechanism of inhibition. The inhibitors were then tested in vitro for their ability to prevent glutamate generation by HIV-infected macrophages, their effect upon macrophage viability, and HIV infection. To validate these findings, glutaminase specific siRNA was tested for its ability to prevent glutamate increase during infection. Our results show that both glutaminase specific small molecule inhibitors and glutaminase specific siRNA were effective at preventing increases in glutamate by HIV-1 infected macrophage. These findings support glutaminase as a potential component of the HAD pathogenic process and identify a possible therapeutic avenue for the treatment of neuroinflammatory states such as HAD.