Improving adoptive T cell therapies of cancer by ectopic expression of miR181a to repress inhibitory phosphatases

Adoptive T cell therapy using antigen-specific T lymphocytes is a powerful immunotherapeutic approach against cancer. Nevertheless, many T cells against tumor-antigens exhibit only weak anti-tumoral response. To overcome this barrier it is necessary to improve the potency and anti-tumoral efficacy of these T cells. Activation and activity of T cells are tightly controlled to inhibit unwanted T cell responses and to reduce the risk of autoimmunity. Both are regulated by extrinsic signals and intrinsic mechanisms which suppress T cell activation. The intrinsic mechanisms include the expression of phosphatases that counteract the activation-inducing kinases. Modifying the expression of these phosphatases allows the targeted modulation of T cell reactivity. MicroRNAs (miRNAs) are regulatory small noncoding RNA molecules that control gene expression by targeting messenger RNAs in a sequence specific manner. Gene-specific silencing plays a key role in diverse biological processes, such as development, differentiation, and functionality. miR181a has been shown to be highly expressed in immature T cells that recognize low-affinity antigens.rnThe present study successfully shows that ectopic expression of miR181a is able to enhance the sensitivity of both murine and human T cells. In CD4+ T helper cells as well as in CD8+ cytotoxic T cells the overexpression of miR181a leads to downregulation of multiple phosphatases involved in the T cell receptor signaling pathway. Overexpression of miR181a in human T cells achieves a co-stimulatory independent activation and has an anti-apoptotic effect on CD4+ T helper cells. Additionally, increasing the amount of miR181a enhances the cytolytic activity of murine CD8+ TCRtg T cells in an antigen-specific manner.rnTo test miR181a overexpressing T cells in vivo, a mouse tumor model using a B cell lymphoma cell line (A20-HA) expressing the Influenza hemagglutinin (Infl.-HA) antigen was established. The expression of model antigens in tumor cell lines enables targeted elimination of tumors using TCRtg T cells. The transfer of miR181a overexpressing Infl.-HA TCRtg CD8+ T cells alone has no positive effect neither on tumor control nor on survival of A20-HA tumor-bearing mice. In contrast, the co-transfer of miR181a overexpressing Infl.-HA TCRtg CD8+ and CD4+ T cells leads to improved tumor control and prolongs survival of A20-HA tumor-bearing mice. This effect is characterized by higher amounts of effector T cells and the expansion of Infl.-HA TCRtg CD8+ T cells.rnAll effects were achieved by changes in expression of several genes including molecules involved in T cell differentiation, activation, and regulation, cytotoxic effector molecules, and receptors important for the homing process of T cells in miR181a overexpressing T cells. The present study demonstrates that miR181a is able to enhance the anti-tumoral response of antigen-specific T cells and is a promising candidate for improving adoptive cell therapy.

Differential inhibition sensitivities of MET mutants to the small molecule inhibitor SU11274

Point mutations emerge as one of the rate-limiting steps in tumor response to small molecule inhibitors of protein kinases. Here we characterized the response of the MET mutated variants, V1110I, V1238I, V1206L and H1112L to the small molecule SU11274. Our results reveal a distinct inhibition pattern of the four mutations with IC(50) values for autophosphorylation inhibition ranging between 0.15 and 1.5muM. Differences were further seen on the ability of SU11274 to inhibit phosphorylation of downstream MET transducers such as AKT, ERK, PLCgamma and STAT3 and a variety of MET-dependent biological endpoints. In all the assays, H1112L was the most sensitive to SU11274, while V1206L was less affected under the used concentration range. The differences in responses to SU11274 are discussed based on a structural model of the MET kinase domain.

Sphingosine kinase 1 is critically involved in nitric oxide-mediated human endothelial cell migration and tube formation

Sphingosine kinases (SKs) convert sphingosine to sphingosine 1-phosphate (S1P), which is a bioactive lipid that regulates a variety of cellular processes including proliferation, differentiation and migration.

Glucocorticoids protect renal mesangial cells from apoptosis by increasing cellular sphingosine-1-phosphate

Neutral ceramidase (NCDase) and sphingosine kinases (SphKs) are key enzymes regulating cellular sphingosine-1-phosphate (S1P) levels. In this study we found that stress factor-induced apoptosis of rat renal mesangial cells was significantly reduced by dexamethasone treatment. Concomitantly, dexamethasone increased cellular S1P levels, suggesting an activation of sphingolipid-metabolizing enzymes. The cell-protective effect of glucocorticoids was reversed by a SphK inhibitor, was completely absent in SphK1-deficient cells, and was associated with upregulated mRNA and protein expression of NCDase and SphK1. Additionally, in vivo experiments in mice showed that dexamethasone also upregulated SphK1 mRNA and activity, and NCDase protein expression in the kidney. Fragments (2285, 1724, and 1126 bp) of the rat NCDase promoter linked to a luciferase reporter were transfected into rat kidney fibroblasts and mesangial cells. There was enhanced NCDase promoter activity upon glucocorticoids treatment that was abolished by the glucocorticoid receptor antagonist RU-486. Single and double mutations of the two putative glucocorticoid response element sites within the promoter reduced the dexamethasone effect, suggesting that both glucocorticoid response elements are functionally active and required for induction. Our study shows that glucocorticoids exert a protective effect on stress-induced mesangial cell apoptosis in vitro and in vivo by upregulating NCDase and SphK1 expression and activity, resulting in enhanced levels of the protective lipid second messenger S1P.

CLEC5A (MDL-1) is a novel PU.1 transcriptional target during myeloid differentiation

C-type lectin domain family 5, member A (CLEC5A), also known as myeloid DNAX activation protein 12 (DAP12)-associating lectin-1 (MDL-1), is a cell surface receptor strongly associated with the activation and differentiation of myeloid cells. CLEC5A associates with its adaptor protein DAP12 to activate a signaling cascade resulting in activation of downstream kinases in inflammatory responses. Currently, little is known about the transcriptional regulation of CLEC5A. We identified CLEC5A as one of the most highly induced genes in a microarray gene profiling experiment of PU.1 restored myeloid PU.1-null cells. We further report that CLEC5A expression is significantly reduced in several myeloid differentiation models upon PU.1 inhibition during monocyte/macrophage or granulocyte differentiation. In addition, CLEC5A mRNA expression was significantly lower in primary acute myeloid leukemia (AML) patient samples than in macrophages and granulocytes from healthy donors. Moreover, we found activation of a CLEC5A promoter reporter by PU.1 as well as in vivo binding of PU.1 to the CLEC5A promoter. Our findings indicate that CLEC5A expression in monocyte/macrophage and granulocytes is regulated by PU.1.

Src inhibitors in lung cancer: current status and future directions

Src tyrosine kinases regulate multiple genetic and signaling pathways involved in the proliferation, survival, angiogenesis, invasion, and migration of various types of cancer cells They are frequently expressed and activated in many cancer types, including lung cancer. Several Src inhibitors, including dasatinib, saracatinib, bosutinib, and KX2-391, are currently being investigated in clinical trials. Preliminary results of the use of single-agent Src inhibitors in unselected patients with lung cancer show that these inhibitors have a favorable safety profile and anticancer activity. Their combination with cytotoxic chemotherapy, other targeted therapy, and radiation therapy is currently being explored. In this review, we summarize the rationale for and the current status of Src inhibitor development and discuss future directions based on emerging preclinical data.

Cancer therapy-associated cardiotoxicity and signaling in the myocardium

The cardiotoxic potential of cytotoxic cancer chemotherapy is well known. Prime examples are the anthracyclines, which are highly efficacious agents for hemopoietic malignancies and solid tumors, but their clinical use is limited primarily by cardiotoxicity. Besides the conventional chemotherapeutics, new cancer drugs were developed in the last decade with the goal to specifically inhibit selected molecular targets such as growth factor receptors or intracellular tyrosine kinases in cancer cells. However, the outcome of combining conventional and newer cancer therapies could have unexpected side effects not anticipated so far and the long-term outcome is not known. Sometimes, however, unexpected side effects also shed light on previously unknown physiological functions. For example, the anti-HER2 cancer therapeutic trastuzumab (Herceptin), which can induce cardiac dysfunction, has demonstrated the importance of the ErbB/neuregulin signaling system in the adult heart. Subsequently, the role of endothelial-myocardial communication in maintaining phenotype and survival of adult cardiomyocytes has increasingly been recognized.

Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis

In development, tissue regeneration or certain diseases, angiogenic growth leads to the expansion of blood vessels and the lymphatic vasculature. This involves endothelial cell proliferation as well as angiogenic sprouting, in which a subset of cells, termed tip cells, acquires motile, invasive behaviour and extends filopodial protrusions. Although it is already appreciated that angiogenesis is triggered by tissue-derived signals, such as vascular endothelial growth factor (VEGF) family growth factors, the resulting signalling processes in endothelial cells are only partly understood. Here we show with genetic experiments in mouse and zebrafish that ephrin-B2, a transmembrane ligand for Eph receptor tyrosine kinases, promotes sprouting behaviour and motility in the angiogenic endothelium. We link this pro-angiogenic function to a crucial role of ephrin-B2 in the VEGF signalling pathway, which we have studied in detail for VEGFR3, the receptor for VEGF-C. In the absence of ephrin-B2, the internalization of VEGFR3 in cultured cells and mutant mice is defective, which compromises downstream signal transduction by the small GTPase Rac1, Akt and the mitogen-activated protein kinase Erk. Our results show that full VEGFR3 signalling is coupled to receptor internalization. Ephrin-B2 is a key regulator of this process and thereby controls angiogenic and lymphangiogenic growth.

Porcine circovirus type 2 DNA influences cytoskeleton rearrangements in plasmacytoid and monocyte-derived dendritic cells

Functional disruption of dendritic cells (DC) is an important strategy for viral pathogens to evade host defences. In this context, porcine circovirus type 2 (PCV2), a single-stranded DNA virus, impairs plasmacytoid DC (pDC) and conventional DC activation by certain viruses or Toll-like receptor (TLR) ligands. This inhibitory capacity is associated with the viral DNA, but the impairment does not affect all signalling cascades; TLR7 ligation by small chemical molecules will still induce interleukin-6 (IL-6) and tumour necrosis factor-α secretion, but not interferon-α or IL-12. In this study, the molecular mechanisms by which silencing occurs were investigated. PP2, a potent inhibitor of the Lyn and Hck kinases, produced a similar profile to the PCV2 DNA interference with cytokine secretion by pDC, efficiently inhibiting cell activation induced through TLR9, but not TLR7, ligation. Confocal microscopy and cytometry analysis strongly suggested that PCV2 DNA impairs actin polymerization and endocytosis in pDC and monocyte-derived DC, respectively. Altogether, this study delineates for the first time particular molecular mechanisms involved in PCV2 interference with DC danger recognition, which may be responsible for the virus-induced immunosuppression observed in infected pigs.

Level of G protein-coupled receptor kinase-2 determines myocardial ischemia/reperfusion injury via pro- and anti-apoptotic mechanisms

Activation of prosurvival kinases and subsequent nitric oxide (NO) production by certain G protein-coupled receptors (GPCRs) protects myocardium in ischemia/reperfusion injury (I/R) models. GPCR signaling pathways are regulated by GPCR kinases (GRKs), and GRK2 has been shown to be a critical molecule in normal and pathological cardiac function.

Regulation of GPCR signaling in hypertension

Hypertension represents a complex, multifactorial disease and contributes to the major causes of morbidity and mortality in industrialized countries: ischemic and hypertensive heart disease, stroke, peripheral atherosclerosis and renal failure. Current pharmacological therapy of essential hypertension focuses on the regulation of vascular resistance by inhibition of hormones such as catecholamines and angiotensin II, blocking them from receptor activation. Interaction of G-protein coupled receptor kinases (GRKs) and regulator of G-protein signaling (RGS) proteins with activated G-protein coupled receptors (GPCRs) effect the phosphorylation state of the receptor leading to desensitization and can profoundly impair signaling. Defects in GPCR regulation via these modulators have severe consequences affecting GPCR-stimulated biological responses in pathological situations such as hypertension, since they fine-tune and balance the major transmitters of vessel constriction versus dilatation, thus representing valuable new targets for anti-hypertensive therapeutic strategies. Elevated levels of GRKs are associated with human hypertensive disease and are relevant modulators of blood pressure in animal models of hypertension. This implies therapeutic perspective in a disease that has a prevalence of 65million in the United States while being directly correlated with occurrence of major adverse cardiac and vascular events. Therefore, therapeutic approaches using the inhibition of GRKs to regulate GPCRs are intriguing novel targets for treatment of hypertension and heart failure.

Gonadotropin-releasing hormone type II antagonist induces apoptosis in MCF-7 and triple-negative MDA-MB-231 human breast cancer cells in vitro and in vivo

Triple-negative breast cancer does not express estrogen and progesterone receptors, and no overexpression/amplification of the HER2-neu gene occurs. Therefore, this subtype of breast cancer lacks the benefits of specific therapies that target these receptors. Today chemotherapy is the only systematic therapy for patients with triple-negative breast cancer. About 50% to 64% of human breast cancers express receptors for gonadotropin-releasing hormone (GnRH), which might be used as a target. New targeted therapies are warranted. Recently, we showed that antagonists of gonadotropin-releasing hormone type II (GnRH-II) induce apoptosis in human endometrial and ovarian cancer cells in vitro and in vivo. This was mediated through activation of stress-induced mitogen-activated protein kinases (MAPKs) p38 and c-Jun N-terminal kinase (JNK), followed by activation of proapoptotic protein Bax, loss of mitochondrial membrane potential, and activation of caspase-3. In the present study, we analyzed whether GnRH-II antagonists induce apoptosis in MCF-7 and triple-negative MDA-MB-231 human breast cancer cells that express GnRH receptors. In addition, we ascertained whether knockdown of GnRH-I receptor expression affects GnRH-II antagonist-induced apoptosis and apoptotic signaling.

On the traces of XPD: cell cycle matters - untangling the genotype-phenotype relationship of XPD mutations

Abstract Mutations in the human gene coding for XPD lead to segmental progeria - the premature appearance of some of the phenotypes normally associated with aging - which may or may not be accompanied by increased cancer incidence. XPD is required for at least three different critical cellular functions: in addition to participating in the process of nucleotide excision repair (NER), which removes bulky DNA lesions, XPD also regulates transcription as part of the general transcription factor IIH (TFIIH) and controls cell cycle progression through its interaction with CAK, a pivotal activator of cyclin dependent kinases (CDKs). The study of inherited XPD disorders offers the opportunity to gain insights into the coordination of important cellular events and may shed light on the mechanisms that regulate the delicate equilibrium between cell proliferation and functional senescence, which is notably altered during physiological aging and in cancer. The phenotypic manifestations in the different XPD disorders are the sum of disturbances in the vital processes carried out by TFIIH and CAK. In addition, further TFIIH- and CAK-independent cellular activities of XPD may also play a role. This, added to the complex feedback networks that are in place to guarantee the coordination between cell cycle, DNA repair and transcription, complicates the interpretation of clinical observations. While results obtained from patient cell isolates as well as from murine models have been elementary in revealing such complexity, the Drosophila embryo has proven useful to analyze the role of XPD as a cell cycle regulator independently from its other cellular functions. Together with data from the biochemical and structural analysis of XPD and of the TFIIH complex these results combine into a new picture of the XPD activities that provides ground for a better understanding of the patophysiology of XPD diseases and for future development of diagnostic and therapeutic tools.

Activation of sphingosine kinase 2 is an endogenous protective mechanism in cerebral ischemia

The two ubiquitously expressed sphingosine kinases (SphK) 1 and 2 are key regulators of the sphingolipid signaling pathway. Despite the formation of an identical messenger, i.e. sphingosine 1-phosphate (S1P), they exert strikingly different functions. Particularly, SphK2 is necessary for the phosphorylation of the sphingosine analog fingolimod (FTY720), which is protective in rodent stroke models. Using gene deficient mice lacking either SphK1 or SphK2, we investigated the role of the two lipid kinases in experimental stroke. We performed 2h transient middle cerebral artery occlusion (tMCAO) and analyzed lesion size and neurological function after 24h. Treatment groups received 1mg/kg FTY720. Neutrophil infiltration, microglia activation, mRNA and protein expression of SphK1, SphK2 and the S1P(1) receptor after tMCAO were studied. Genetic deletion of SphK2 but not SphK1 increased ischemic lesion size and worsened neurological function after tMCAO. The protective effect of FTY720 was conserved in SphK1(-/-) mice but not in SphK2(-/-) mice. This suggests that SphK2 activity is an important endogenous protective mechanism in cerebral ischemia and corroborates that the protective effect of FTY720 is mediated via phospho-FTY720.

The sphingosine kinase 1 and S1P1 axis specifically counteracts LPS-induced IL-12p70 production in immune cells of the spleen

Sphingosine-1-phosphate (S1P) has been implicated in angiogenesis, inflammation, cancerogenesis, neurological excitability and immune regulation and is synthesized by two different sphingosine kinases (SphK). It was suggested that mice lacking the gene for SphK1 exhibit no obvious phenotype, because SphK2 compensates for its absence. However, recent investigations revealed that under challenge SphK1 contributed to pro-inflammatory processes favoring Th2 and Th17 rather than Th1-type reactions. To investigate the immune modulatory role of SphK1 as opposed to SphK2 specifically for the Th1 propagating IL-12p70 we compared WT and SphK1(-/-) splenocytes and Flt3-ligand differentiated BMCs of WT and SphK1(-/-), representing dendritic cells as major producers of IL-12p70, incubated with LPS. We determined the impact on IL-12p70 in comparison to other inflammatory cytokines, and on DC and macrophage surface marker expression, SphK mRNA, protein expression and enzymatic activity in splenocytes. Our data demonstrated that SphK1 deficiency enhanced LPS-induced IL-12p70 production although SphK2 was present. To further characterize SphK1-dependent IL-12p70 regulation we exogenously applied S1P, SEW2871 and the new potent S1P1 agonist CYM5442. Both S1P and S1P1-specific analogs fully compensated the increase of IL-12p70 production in SphK1-deficient splenocytes. The use of pertussis toxin, to block G(i)-coupled signaling downstream of S1P1, again increased IL-12p70 and neglected the compensation achieved by addition of S1P and S1P1 agonists pointing on the importance of this specific S1P-receptor. Given that, in parallel to a prominent IL-12p35 increase following LPS stimulation, LPS also enhanced SphK expression and total SphK activity, we concluded that SphK1-derived S1P acting via S1P1 is a major mechanism of this negative IL-12p70 feedback loop, which did not affect other cytokines. Moreover, our data showed that SphK2 activity failed to compensate for SphK1 deficiency. These findings clearly point to a divergent and cytokine-specific impact of immune cell SphK1 and SphK2 in chronic inflammation and cancer.