Therapeutically targeting the unique disease landscape of pediatric high-grade gliomas.

Pediatric high-grade gliomas (pHGG) are a rare yet devastating malignancy of the central nervous system's glial support cells, affecting children, adolescents, and young adults. Tumors of the central nervous system account for the leading cause of pediatric mortality of which high-grade gliomas present a significantly grim prognosis. While the past few decades have seen many pediatric cancers experiencing significant improvements in overall survival, the prospect of survival for patients diagnosed with pHGGs has conversely remained unchanged. This can be attributed in part to tumor heterogeneity and the existence of the blood-brain barrier. Advances in discovery research have substantiated the existence of unique subgroups of pHGGs displaying alternate responses to different therapeutics and varying degrees of overall survival. This highlights a necessity to approach discovery research and clinical management of the disease in an alternative subtype-dependent manner. This review covers traditional approaches to the therapeutic management of pHGGs, limitations of such methods and emerging alternatives. Novel mutations which predominate the pHGG landscape are highlighted and the therapeutic potential of targeting them in a subtype specific manner discussed. Collectively, this provides an insight into issues in need of transformative progress which arise during the management of pHGGs.

Integrin-linked kinase expression in myeloid cells promotes colon tumorigenesis.

Colorectal cancer (CRC) is one of the most common forms of cancer worldwide and treatment options for advanced CRC, which has a low 5-year survival rate, remain limited. Integrin-linked kinase (ILK), a multifunctional, scaffolding, pseudo-kinase regulating many integrin-mediated cellular processes, is highly expressed in many cancers. However, the role of ILK in cancer progression is yet to be fully understood. We have previously uncovered a pro-inflammatory role for myeloid-specific ILK in dextran sodium sulfate (DSS)-induced colitis. To establish a correlation between chronic intestinal inflammation and colorectal cancer (CRC), we investigated the role of myeloid-ILK in mouse models of CRC. When myeloid-ILK deficient mice along with the WT control mice were subjected to colitis-associated and APCmin/+-driven CRC, tumour burden was reduced by myeloid-ILK deficiency in both models. The tumour-promoting phenotype of macrophages, M2 polarization, in vitro was impaired by the ILK deficiency and the number of M2-specific marker CD206-expressing tumour-associated macrophages (TAMs) in vivo were significantly diminished in myeloid-ILK deficient mice. Myeloid-ILK deficient mice showed enhanced tumour infiltration of CD8+ T cells and reduced tumour infiltration of FOXP3+ T cells in colitis-associated and APCmin/+-driven CRC, respectively, with an overall elevated CD8+/FOXP3+ ratio suggesting an anti-tumour immune phenotypes. In patient CRC tissue microarrays we observed elevated ILK+ myeloid (ILK+ CD11b+) cells in tumour sections compared to adjacent normal tissues, suggesting a conserved role for myeloid-ILK in CRC development in both human and animal models. This study identifies myeloid-specific ILK expression as novel driver of CRC, which could be targeted as a potential therapeutic option for advanced disease.

The protein kinase R modifies gut physiology to limit colitis.

Here we investigate the function of the innate immune molecule protein kinase R (PKR) in intestinal inflammation. To model a colitogenic role of PKR, we determine the physiological response to dextran sulfate sodium (DSS) of wild-type and two transgenic mice strains mutated to express either a kinase-dead PKR or to ablate expression of the kinase. These experiments recognize kinase-dependent and -independent protection from DSS-induced weight loss and inflammation, against a kinase-dependent increase in the susceptibility to DSS-induced injury. We propose these effects arise through PKR-dependent alteration of gut physiology, evidenced as altered goblet cell function and changes to the gut microbiota at homeostasis that suppresses inflammasome activity by controlling autophagy. These findings establish that PKR functions as both a protein kinase and a signaling molecule in instituting immune homeostasis in the gut.

Integrin-Linked Kinase Expression Characterizes the Immunosuppressive Tumor Microenvironment in Colorectal Cancer and Regulates PD-L1 Expression and Immune Cell Cytotoxicity.

Integrin-linked kinase (ILK) has been implicated as a molecular driver and mediator in both inflammation and tumorigenesis of the colon. However, a role for ILK in the tumor microenvironment (TME) and immune evasion has not been investigated. Here, we show a correlation of ILK expression with the immunosuppressive TME and cancer prognosis. We also uncover a role for ILK in the regulation of programmed death-ligand 1 (PD-L1) expression and immune cell cytotoxicity. Interrogation of web-based data-mining platforms, showed upregulation of ILK expression in tumors and adjacent-non tumor tissue of colorectal cancer (CRC) associated with poor survival and advanced stages. ILK expression was correlated with cancer-associated fibroblast (CAFs) and immunosuppressive cell infiltration including regulatory T cells (Treg) and M2 macrophages (M2) in addition to their gene markers. ILK expression was also significantly correlated with the expression of different cytokines and chemokines. ILK expression showed pronounced association with different important immune checkpoints including PD-L1. Deletion of the ILK gene in PD-L1 positive CRC cell lines using a doxycycline inducible-CRISPR/Cas9, resulted in suppression of both the basal and IFNγ-induced PD-L1 expression via downregulating NF-κB p65. This subsequently sensitized the CRC cells to NK92 immune cell cytotoxicity. These findings suggest that ILK can be used as a biomarker for prognosis and immune cell infiltration in colon cancer. Moreover, ILK could provide a therapeutic target to prevent immune evasion mediated by the expression of PD-L1.

A Potential Role for Integrin-Linked Kinase in Colorectal Cancer Growth and Progression via Regulating Senescence and Immunity.

Integrin-linked kinase (ILK) has been implicated as a molecular driver and mediator in both inflammation and tumorigenesis of the colon. ILK functions as an adaptor and mediator protein linking the extracellular matrix with downstream signaling pathways. ILK is broadly expressed in many human tissues and cells. It is also overexpressed in many cancers, including colorectal cancer (CRC). Inflammation, as evidenced by inflammatory bowel disease (IBD), is one of the highest risk factors for initiating CRC. This has led to the hypothesis that targeting ILK therapeutically could have potential in CRC, as it regulates different cellular processes associated with CRC development and progression as well as inflammation in the colon. A number of studies have indicated an ILK function in senescence, a cellular process that arrests the cell cycle while maintaining active metabolism and transcription. Senescent cells produce different secretions collectively known as the senescence-associated secretory phenotype (SASP). The SASP secretions influence infiltration of different immune cells, either positively for clearing senescent cells or negatively for promoting tumor growth, reflecting the dual role of senescence in cancer. However, a role for ILK in senescence and immunity in CRC remains to be determined. In this review, we discuss the possible role for ILK in senescence and immunity, paying particular attention to the relevance of ILK in CRC. We also examine how activating Toll-like receptors (TLRs) and their agonists in CRC could trigger immune responses against cancer, as a combination therapy with ILK inhibition.

Mechanisms and consequences of constitutive activation of integrin-linked kinase in acute myeloid leukemia.

Integrin-linked kinase (ILK) has emerged as a critical adaptor and mediator protein in cell signaling pathways that is commonly deregulated in acute myeloid leukemia (AML). This has led to the expectation that therapeutic targeting of ILK may be a useful option in treating leukemia. Although ILK can regulate many cellular processes, including cell differentiation, survival, migration, apoptosis and production of pro-inflammatory cytokines, its role in promoting AML is still unclear. However, its ability to mediate phosphorylation and regulate the important hematopoietic stem cell regulators protein kinase B (AKT) and glycogen synthase kinase-3ß supports ILK as an attractive target for the development of novel anticancer therapeutics. In this review, we summarize the existing knowledge of ILK signaling and its impact on cytokines, paying particular attention to the relevance of ILK signaling in AML. We also discuss the rationale for targeting ILK in the treatment of AML and conclude with perspectives on the future of ILK-targeted therapy in AML.

Integrin-Linked Kinase Expression in Myeloid Cells Promotes Inflammatory Signaling during Experimental Colitis.

The pathology of inflammatory bowel diseases is driven by the inflammatory signaling pathways associated with mucosal epithelial damage. Myeloid cells are known to play an essential role in mediating epithelial inflammatory responses during injury. However, the precise role of these cells in stimulating intestinal inflammation and the subsequent tissue damage is unclear. In this article, we show that expression of integrin-linked kinase (ILK) in myeloid cells is critical for the epithelial inflammatory signaling during colitis induced by dextran sodium sulfate. Myeloid ILK (M-ILK) deficiency significantly ameliorates the pathology of experimental colitis. In response to dextran sodium sulfate, colonic infiltration of neutrophils and inflammatory cytokine production are impaired in M-ILK-deficient mice, and activation of epithelial NF-κB and PI3K signaling pathways are restricted by the M-ILK deficiency. In contrast, reduced epithelial damage in M-ILK-deficient mice is correlated with elevated levels of epithelial Stat3 activation and proliferation. Moreover, M-ILK-dependent inflammatory signaling in the mucosal epithelium can be therapeutically targeted by the pharmacological inhibition of ILK during experimental colitis. Collectively, these findings identify M-ILK as a critical regulator of epithelial inflammatory signaling pathways during colitis and, as a consequence, targeting M-ILK could provide therapeutic benefit.

Transcriptional Activation of Inflammatory Genes: Mechanistic Insight into Selectivity and Diversity.

Acute inflammation, an integral part of host defence and immunity, is a highly conserved cellular response to pathogens and other harmful stimuli. An inflammatory stimulation triggers transcriptional activation of selective pro-inflammatory genes that carry out specific functions such as anti-microbial activity or tissue healing. Based on the nature of inflammatory stimuli, an extensive exploitation of selective transcriptional activations of pro-inflammatory genes is performed by the host to ensure a defined inflammatory response. Inflammatory signal transductions are initiated by the recognition of inflammatory stimuli by transmembrane receptors, followed by the transmission of the signals to the nucleus for differential gene activations. The differential transcriptional activation of pro-inflammatory genes is precisely controlled by the selective binding of transcription factors to the promoters of these genes. Among a number of transcription factors identified to date, NF-κB still remains the most prominent and studied factor for its diverse range of selective transcriptional activities. Differential transcriptional activities of NF-κB are dictated by post-translational modifications, specificities in dimer formation, and variability in activation kinetics. Apart from the differential functions of transcription factors, the transcriptional activation of selective pro-inflammatory genes is also governed by chromatin structures, epigenetic markers, and other regulators as the field is continuously expanding.

Integrin-linked kinase modulates lipopolysaccharide- and Helicobacter pylori-induced nuclear factor κB-activated tumor necrosis factor-α production via regulation of p65 serine 536 phosphorylation.

Integrin-linked kinase (ILK) is a ubiquitously expressed and highly conserved serine-threonine protein kinase that regulates cellular responses to a wide variety of extracellular stimuli. ILK is involved in cell-matrix interactions, cytoskeletal organization, and cell signaling. ILK signaling has also been implicated in oncogenesis and progression of cancers. However, its role in the innate immune system remains unknown. Here, we show that ILK mediates pro-inflammatory signaling in response to lipopolysaccharide (LPS). Pharmacological or genetic inhibition of ILK in mouse embryonic fibroblasts and macrophages selectively blocks LPS-induced production of the pro-inflammatory cytokine tumor necrosis factor α (TNF-α). ILK is required for LPS-induced activation of nuclear factor κB (NF-κB) and transcriptional induction of TNF-α. The modulation of LPS-induced TNF-α synthesis by ILK does not involve the classical NF-κB pathway, because IκB-α degradation and p65 nuclear translocation are both unaffected by ILK inhibition. Instead, ILK is involved in an alternative activation of NF-κB signaling by modulating the phosphorylation of p65 at Ser-536. Furthermore, ILK-mediated alternative NF-κB activation through p65 Ser-536 phosphorylation also occurs during Helicobacter pylori infection in macrophages and gastric cancer cells. Moreover, ILK is required for H. pylori-induced TNF-α secretion in macrophages. Although ILK-mediated phosphorylation of p65 at Ser-536 is independent of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway during LPS stimulation, upon H. pylori infection this event is dependent on the PI3K/Akt pathway. Our findings implicate ILK as a critical regulatory molecule for the NF-κB-mediated pro-inflammatory signaling pathway, which is essential for innate immune responses against pathogenic microorganisms.

Identification of NURR1 as a mediator of MIF signaling during chronic arthritis: effects on glucocorticoid-induced MKP1.

Elucidation of factors regulating glucocorticoid (GC) sensitivity is required for the development of "steroid-sparing" therapies for chronic inflammatory diseases, including rheumatoid arthritis (RA). Accumulating evidence suggests that macrophage migration inhibitory factor (MIF) counterregulates the GC-induction of anti-inflammatory mediators, including mitogen-activated protein kinase phosphatase 1 (MKP1), a critical mitogen-activated protein kinase signaling inhibitor. This observation has yet to be extended to human disease; the molecular mechanisms remain unknown. We investigated NURR1, a GC-responsive transcription factor overexpressed in RA, as a MIF signaling target. We reveal abrogation by recombinant MIF (rMIF) of GC-induced MKP1 expression in RA fibroblast-like synoviocytes (FLS). rMIF enhanced NURR1 expression, artificial NBRE (orphan receptor DNA-binding site) reporter transactivation, and reversed GC-inhibition of NURR1. NURR1 expression was reduced during experimental arthritis in MIF-/- synovium, and silencing MIF reduced RA FLS NURR1 mRNA. Consistent with NBRE identification on the MKP1 gene, MKP1 mRNA was reduced in FLS that ectopically express NURR1, and silencing NURR1 enhanced MKP1 mRNA in RA FLS. rMIF enhanced NBRE binding on the MKP1 gene, and the absence of the NBRE prevented NURR1-repressive effects on basal and GC-induced MKP1 transactivation. This study defines NURR1 as a novel MIF target in chronic inflammation and demonstrates a role for NURR1 in regulating the anti-inflammatory mediator, MKP1. We propose a MIF-NURR1 signaling axis as a regulator of the GC sensitivity of MKP1.

Import of nuclear-encoded mitochondrial proteins: a cotranslational perspective.

A growing amount of evidence suggests that the cytosolic translation of nuclear-encoded mitochondrial proteins and their subsequent import into mitochondria are tightly coupled in a process termed cotranslational import. In addition to the original posttranslational view of mitochondrial protein import, early literature also provides both in vitro and in vivo experimental evidence supporting the simultaneous existence of a cotranslational protein-import mechanism in mitochondria. Recent investigations have started to reveal the cotranslational import mechanism which is initiated by transporting either a translation complex or a translationally competent mRNA encoding a mitochondrial protein to the mitochondrial surface. The intracellular localization of mRNA to the mitochondrial surface has emerged as the latest addition to our understanding of mitochondrial biogenesis. It is mediated by targeting elements within the mRNA molecule in association with potential mRNA-binding proteins.

IAP antagonists target cIAP1 to induce TNFalpha-dependent apoptosis.

XIAP prevents apoptosis by binding to and inhibiting caspases, and this inhibition can be relieved by IAP antagonists, such as Smac/DIABLO. IAP antagonist compounds (IACs) have therefore been designed to inhibit XIAP to kill tumor cells. Because XIAP inhibits postmitochondrial caspases, caspase 8 inhibitors should not block killing by IACs. Instead, we show that apoptosis caused by an IAC is blocked by the caspase 8 inhibitor crmA and that IAP antagonists activate NF-kappaB signaling via inhibtion of cIAP1. In sensitive tumor lines, IAP antagonist induced NF-kappaB-stimulated production of TNFalpha that killed cells in an autocrine fashion. Inhibition of NF-kappaB reduced TNFalpha production, and blocking NF-kappaB activation or TNFalpha allowed tumor cells to survive IAC-induced apoptosis. Cells treated with an IAC, or those in which cIAP1 was deleted, became sensitive to apoptosis induced by exogenous TNFalpha, suggesting novel uses of these compounds in treating cancer.

Filamin repeat segments required for photosensory signalling in Dictyostelium discoideum.

BACKGROUND: Filamin is an actin binding protein which is ubiquitous in eukaryotes and its basic structure is well conserved - an N-terminal actin binding domain followed by a series of repeated segments which vary in number in different organisms. D. discoideum is a well established model organism for the study of signalling pathways and the actin cytoskeleton and as such makes an excellent organism in which to study filamin. Ddfilamin plays a putative role as a scaffolding protein in a photosensory signalling pathway and this role is thought to be mediated by the unusual repeat segments in the rod domain. RESULTS: To study the role of filamin in phototaxis, a filamin null mutant, HG1264, was transformed with constructs each of which expressed wild type filamin or a mutant filamin with a deletion of one of the repeat segments. Transformants expressing the full length filamin to wild type levels completely rescued the phototaxis defect in HG1264, however if filamin was expressed at lower than wild type levels the phototaxis defect was not restored. The transformants lacking any one of the repeat segments 2-6 retained defective phototaxis and thermotaxis phenotypes, whereas transformants expressing filaminDelta1 exhibited a range of partial complementation of the phototaxis phenotype which was related to expression levels. Immunofluorescence microscopy showed that filamin lacking any of the repeat segments still localised to the same actin rich areas as wild type filamin. Ddfilamin interacts with RasD and IP experiments demonstrated that this interaction did not rely upon any single repeat segment or the actin binding domain. CONCLUSION: This paper demonstrates that wild type levels of filamin expression are essential for the formation of functional photosensory signalling complexes and that each of the repeat segments 2-6 are essential for filamins role in phototaxis. By contrast, repeat segment 1 is not essential provided the mutated filamin lacking repeat segment 1 is expressed at a high enough level. The defects in photo/thermosensory signal transduction caused by the absence of the repeats are due neither to mislocalisation of filamin nor to the loss of RasD recruitment to the previously described photosensory signalling complex.

Diverse cytopathologies in mitochondrial disease are caused by AMP-activated protein kinase signaling.

The complex cytopathology of mitochondrial diseases is usually attributed to insufficient ATP. AMP-activated protein kinase (AMPK) is a highly sensitive cellular energy sensor that is stimulated by ATP-depleting stresses. By antisense-inhibiting chaperonin 60 expression, we produced mitochondrially diseased strains with gene dose-dependent defects in phototaxis, growth, and multicellular morphogenesis. Mitochondrial disease was phenocopied in a gene dose-dependent manner by overexpressing a constitutively active AMPK alpha subunit (AMPKalphaT). The aberrant phenotypes in mitochondrially diseased strains were suppressed completely by antisense-inhibiting AMPKalpha expression. Phagocytosis and macropinocytosis, although energy consuming, were unaffected by mitochondrial disease and AMPKalpha expression levels. Consistent with the role of AMPK in energy homeostasis, mitochondrial "mass" and ATP levels were reduced by AMPKalpha antisense inhibition and increased by AMPKalphaT overexpression, but they were near normal in mitochondrially diseased cells. We also found that 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside, a pharmacological AMPK activator in mammalian cells, mimics mitochondrial disease in impairing Dictyostelium phototaxis and that AMPKalpha antisense-inhibited cells were resistant to this effect. The results show that diverse cytopathologies in Dictyostelium mitochondrial disease are caused by chronic AMPK signaling not by insufficient ATP.

Import-associated translational inhibition: novel in vivo evidence for cotranslational protein import into Dictyostelium discoideum mitochondria.

To investigate protein import into the mitochondria of Dictyostelium discoideum, green fluorescent protein (GFP) was fused as a reporter protein either to variable lengths of the N-terminal region of chaperonin 60 (the first 23, 40, 80, 97, and 150 amino acids) or to the mitochondrial targeting sequence of DNA topoisomerase II. The fusion proteins were expressed in AX2 cells under the actin-15 promoter. Fluorescence images of GFP transformants confirmed that Dictyostelium chaperonin 60 is a mitochondrial protein. The level of the mitochondrially targeted GFP fusion proteins was unexpectedly much lower than the nontargeted (cytoplasmic) forms. The distinction between targeted and nontargeted protein activities was investigated at both the transcriptional and translational levels in vivo. We found that targeting GFP to the mitochondria results in reduced levels of the fusion protein even though transcription of the fusion gene and the stability of the protein are unaffected. [(35)S]methionine labeling and GFP immunoprecipitation confirmed that mitochondrially targeted GFP is translated at much slower rates than nontargeted GFP. The results indicate a novel phenomenon, import-associated translational inhibition, whereby protein import into the mitochondria limits the rate of translation. The simplest explanation for this is that import of the GFP fusion proteins occurs cotranslationally, i.e., protein synthesis and import into mitochondria are coupled events. Consistent with cotranslational import, Northern analysis showed that the GFP mRNA is associated with isolated mitochondria. This association occurred regardless of whether the GFP was fused to a mitochondrial leader peptide. However, the presence of an import-competent leader peptide stabilized the mRNA-mitochondria association, rendering it more resistant to extensive EDTA washing. In contrast with GFP, the mRNA of another test protein, aequorin, did not associate with the mitochondria, and its translation was unaffected by import of the encoded polypeptide into the mitochondria.