Upregulation of miRNA-155 promotes tumour angiogenesis by targeting VHL and is associated with poor prognosis and triple-negative breast cancer.

MicroRNA-155 (miR-155) is frequently upregulated in various types of human cancer; however, its role in cancer angiogenesis remains unknown. Here, we demonstrate the role of miR-155 in angiogenesis through targeting von Hippel-Lindau (VHL) tumour suppressor in breast cancer. Ectopic expression of miR-155 induced whereas knockdown of miR-155 inhibited human umbilical vein endothelial cell network formation, proliferation, invasion and migration. Furthermore, mammary fat pad xenotransplantation of ectopically expressed miR-155 resulted in extensive angiogenesis, proliferation, tumour necrosis and recruitment of pro-inflammatory cells such as tumour-associated macrophages. Expression of VHL abrogated these miR-155 effects. Moreover, miR-155 expression inversely correlates with VHL expression level and is associated with late-stage, lymph node metastasis and poor prognosis, as well as triple-negative tumour in breast cancer. These findings indicate that miR-155 has a pivotal role in tumour angiogenesis by downregulation of VHL, and provide a basis for miR-155-expressing tumours to embody an aggressive malignant phenotype, and therefore miR-155 is an important therapeutic target in breast cancer.

Novel role of Stat1 in the development of docetaxel resistance in prostate tumor cells.

A major obstacle for clinicians in the treatment of advanced prostate cancer is the inevitable progression to chemoresistance, especially to docetaxel. It is essential to understand the molecular events that lead to docetaxel resistance in order to identify means to prevent or interfere with chemoresistance. In initial attempts to detect these events, we analysed genomic differences between non-resistant and docetaxel-resistant prostate tumor cells and, of the genes modulated by docetaxel treatment, we observed Stat1 and clusterin gene expression heightened in the resistant phenotype. In this study, we provide biochemical and biological evidence that these two gene products are related. Stat1 and clusterin protein expression was induced upon docetaxel treatment of DU145 cells and highly overexpressed in the docetaxel-resistant DU145 cells (DU145-DR). The increase in total Stat1 corresponded to an increase in phosphorylated Stat1. Interestingly, there was no detectable difference between DU145 and DU145-DR cells expression of total Stat3 and phosphorylated Stat3. Treatment of DU145-DR cells with small interfering RNA targeted for Stat1 not only resulted in the knockdown of Stat1 expression, but it also caused the inhibition of clusterin expression. Thus, Stat1 appears to play a key role in the regulation of clusterin. Remarkably, inhibition of Stat1 or clusterin expression resulted in the re-sensitization of DU145-DR cells to docetaxel. These results offer the first evidence that Stat1, and its subsequent regulation of clusterin, are essential for docetaxel resistance in prostate cancer. Targeting this pathway could be a potential therapeutic means for intervention of docetaxel resistance.

Immune-type receptor genes in zebrafish share genetic and functional properties with genes encoded by the mammalian leukocyte receptor cluster.

An extensive, highly diversified multigene family of novel immune-type receptor (nitr) genes has been defined in Danio rerio (zebrafish). The genes are predicted to encode type I transmembrane glycoproteins consisting of extracellular variable (V) and V-like C2 (V/C2) domains, a transmembrane region and a cytoplasmic tail. All of the genes examined encode immunoreceptor tyrosine-based inhibition motifs in the cytoplasmic tail. Radiation hybrid panel mapping and analysis of a deletion mutant line (b240) indicate that a minimum of approximately 40 nitr genes are contiguous in the genome and span approximately 0.6 Mb near the top of zebrafish linkage group 7. One flanking region of the nitr gene complex shares conserved synteny with a region of mouse chromosome 7, which shares conserved synteny with human 19q13.3-q13.4 that encodes the leukocyte receptor cluster. Antibody-induced crosslinking of Nitrs that have been introduced into a human natural killer cell line inhibits the phosphorylation of mitogen-activated protein kinase that is triggered by natural killer-sensitive tumor target cells. Nitrs likely represent intermediates in the evolution of the leukocyte receptor cluster.

Cooperative regulation of Mcl-1 by Janus kinase/stat and phosphatidylinositol 3-kinase contribute to granulocyte-macrophage colony-stimulating factor-delayed apoptosis in human neutrophils.

Polymorphonuclear neutrophils (PMN) are phagocytic cells constitutively programmed for apoptotic cell death. Exposure to GM-CSF delays apoptosis as measured by annexin-V staining and cell morphological change. We found that STAT5B, STAT1, and STAT3 DNA-binding activity was induced by GM-CSF. We also detected activation of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway after GM-CSF treatment which was inhibited by treatment with the PI 3-kinase inhibitors, wortmannin and LY294002. We investigated whether STAT or PI 3-kinase activity was necessary for the pro-survival response of GM-CSF in PMN. Exposure of PMN to GM-CSF in the presence of either AG-490, antisense STAT3 oligonucleotides, or wortmannin resulted in a partial inhibition of GM-CSF-mediated pro-survival activity. GM-CSF induced a time-dependent increase in the mRNA and protein expression of the anti-apoptotic Bcl-2-family protein, Mcl-1. We examined the hypothesis that Janus kinase/STAT and PI 3-kinase regulation of Mcl-1 contributed to GM-CSF-delayed apoptosis. Using either AG-490 or wortmannin alone, we observed a dose-dependent inhibition of GM-CSF-induced Mcl-1 expression. Using suboptimal doses of AG-490 and wortmannin, we found that both drugs together had an additive effect on delayed apoptosis and Mcl-1 expression. These data suggest that cooperative regulation of Mcl-1 by the Janus kinase/STAT and PI 3-kinase pathways contribute to GM-CSF-delayed apoptosis.

IL-2 induces the association of IL-2Rbeta, lyn, and MAP kinase ERK-1 in human neutrophils.

IL-2, first identified as a T cell growth factor, has been proven to activate many cell types including polymorphonuclear neutrophils (PMN3). However, the mechanisms involved in PMN activation, especially the signaling pathways used by the IL-2R, are currently unknown. Here we demonstrate that IL-2 has the ability to induce protein tyrosine kinases in human PMN, and we provide the first evidence that lyn kinase is activated and physically associated with MAP kinase/ERK1. Co-immunoprecipitation experiments with anti-IL-2Rbeta and Western blotting with anti-p53/56lym revealed that lyn protein was present in IL-2R precipitates and that the association of lyn with IL-2Rbeta was markedly elevated by IL-2 stimulation. Furthermore the activity of lyn kinase, evaluated by an in vitro kinase assay with enolase as a substrate, increased following IL-2 stimulation. Another important finding was that, upon IL-2 activation, MAPK/ERK1 was also phosphorylated in PMN. A direct association between lyn and ERK1 was initially demonstrated by co-immunoprecipitation/Western blotting and then definitively proven by the use of a GST-ERK1 fusion protein. We showed that ERK1 binds lyn only in IL-2 stimulated PMN, but not in unstimulated PMN. These results suggest that IL-2 can promote the association of lyn protein tyrosine kinase with IL-2Rbeta as well as the direct binding of MAPK/ERK1 to lyn. The signaling pathway utilized by human PMN in response to IL-2 may thus involve the association of lyn with IL-2Rbeta and the activation process also triggers the recruitment and activation of a specific ERK.

Direct tumor lysis by NK cells uses a Ras-independent mitogen-activated protein kinase signal pathway.

Destruction of tumor cells is a key function of lymphocytes, but the molecular processes driving it are unclear. Analysis of signal molecules indicated that mitogen-activated protein kinase (MAPK)/extracellular regulated kinase 2 critically controlled lytic function in human NK cells. We now have evidence to indicate that target ligation triggers a Ras-independent MAPK pathway that is required for lysis of the ligated tumor cell. Target engagement caused NK cells to rapidly activate MAPK within 5 min, and PD098059 effectively blocked both MAPK activation and tumoricidal function in NK cells. Target engagement also rapidly activated Ras, detected as active Ras-GTP bound to GST-Raf-RBD, a GST fusion protein linked to the Raf protein fragment containing the Ras-GTP binding domain. However, Ras inactivation by pharmacological disruption with the farnesyl transferase inhibitor, FTI-277, had no adverse effect on the ability of NK cells to lyse tumor cells or to express MAPK activation upon target conjugation. Notably, MAPK inactivation with PD098059, but not Ras inactivation with FTI-277, could interfere with perforin and granzyme B polarization within NK cells toward the contacted target cell. Using vaccinia delivery of N17 Ras into NK cells, we demonstrated that IL-2 activated a Ras-dependent MAPK pathway, while target ligation used a Ras-independent MAPK pathway to trigger lysis in NK cells.

Chronic neutropenia mediated by fas ligand.

Chronic neutropenia, often associated with rheumatoid arthritis, is a characteristic finding in large granular lymphocyte (LGL) leukemia. The mechanism of neutropenia is not known. Normal neutrophil survival is regulated by the Fas-Fas ligand apoptotic system. We hypothesized that neutropenia in LGL leukemia is mediated by dysregulated expression of Fas ligand. Levels of Fas ligand in serum samples from patients with LGL leukemia were measured with a Fas ligand enzyme-linked immunosorbent assay. The effects of serum from patients with LGL leukemia on apoptosis of normal neutrophils were determined by flow cytometry and morphologic assessment. High levels of circulating Fas ligand were detected in 39 of 44 serum samples from patients with LGL leukemia. In contrast, Fas ligand was undetectable in 10 samples from healthy donors. Serum from the patients triggered apoptosis of normal neutrophils that depended partly on the Fas pathway. Resolution of neutropenia was associated with disappearance or marked reduction in Fas ligand levels in 10 of 11 treated patients. These data suggest that high levels of Fas ligand are a pathogenetic mechanism in human disease. (Blood. 2000;95:3219-3222)

Retinoblastoma protein activation of interleukin 8 expression inhibits tumor cell survival in nude mice.

Loss of retinoblastoma protein (Rb) has been implicated in the formation of a variety of human malignancies. Restoration of Rb expression in the cell lines representing these tumors eliminates or significantly reduces tumorigenicity in nude mice, but the mechanism for this Rb effect is unknown. Results from this study indicated that Rb expression reduced tumor cell survival in nude mice by dramatically enhancing interleukin 8 (IL-8) secretion. IL-8 secreted by the Rb-transformed cells attracted neutrophils in vitro and tumor-infiltrating neutrophils in vivo, which is consistent with the Rb-mediated tumor regression being dependent on IL-8. The apparent, contradictory roles of IL-8 as a protumorigenic and antitumorigenic cytokine are discussed.

Pivotal role of phosphoinositide-3 kinase in regulation of cytotoxicity in natural killer cells.

The mitogen-activated protein kinase-extracellular signal-regulated kinase signaling element (MAPK-ERK) plays a critical role in natural killer (NK) cell lysis of tumor cells, but its upstream effectors were previously unknown. We show that inhibition of phosphoinositide-3 kinase (PI3K) in NK cells blocks p21-activated kinase 1 (PAK1), MAPK kinase (MEK) and ERK activation by target cell ligation, interferes with perforin and granzyme B movement toward target cells and suppresses NK cytotoxicity. Dominant-negative N17Rac1 and PAK1 mimic the suppressive effects of PI3K inhibitors, whereas constitutively active V12Rac1 has the opposite effect. V12Rac1 restores the activity of downstream effectors and lytic function in LY294002- or wortmannin-treated, but not PD98059-treated, NK cells. These results document a specific PI3K-->Rac1-->PAK1-->MEK-->ERK pathway in NK cells that effects lysis.

Requirement for Ras/Rac1-mediated p38 and c-Jun N-terminal kinase signaling in Stat3 transcriptional activity induced by the Src oncoprotein.

Signal transducers and activators of transcription (STATs) are transcription factors that mediate normal biologic responses to cytokines and growth factors. However, abnormal activation of certain STAT family members, including Stat3, is increasingly associated with oncogenesis. In fibroblasts expressing the Src oncoprotein, activation of Stat3 induces specific gene expression and is required for cell transformation. Although the Src tyrosine kinase induces constitutive Stat3 phosphorylation on tyrosine, activation of Stat3-mediated gene regulation requires both tyrosine and serine phosphorylation of Stat3. We investigated the signaling pathways underlying the constitutive Stat3 activation in Src oncogenesis. Expression of Ras or Rac1 dominant negative protein blocks Stat3-mediated gene regulation induced by Src in a manner consistent with dependence on p38 and c-Jun N-terminal kinase (JNK). Both of these serine/threonine kinases and Stat3 serine phosphorylation are constitutively induced in Src-transformed fibroblasts. Furthermore, inhibition of p38 and JNK activities suppresses constitutive Stat3 serine phosphorylation and Stat3-mediated gene regulation. In vitro kinase assays with purified full-length Stat3 as the substrate show that both JNK and p38 can phosphorylate Stat3 on serine. Moreover, inhibition of p38 activity and thus of Stat3 serine phosphorylation results in suppression of transformation by v-Src but not v-Ras, consistent with a requirement for Stat3 serine phosphorylation in Src transformation. Our results demonstrate that Ras- and Rac1-mediated p38 and JNK signals are required for Stat3 transcriptional activity induced by the Src oncoprotein. These findings delineate a network of tyrosine and serine/threonine kinase signaling pathways that converge on Stat3 in the context of oncogenesis.

Retinoblastoma protein expression leads to reduced Oct-1 DNA binding activity and enhances interleukin-8 expression.

Tumor cell lines with a defective retinoblastoma gene are unable to transcribe the HLA class II genes in response to IFN-gamma treatment, and reconstitution of functional Rb rescues IFN-gamma-induced class II gene expression. However, the molecular mechanism of Rb rescue of the class II genes is unknown. We have examined the effect of Rb expression on the activation of the promoter for HLA-DRA, the prototype class II gene. Oct-1, a POU domain transcription factor, was identified as a repressor of HLA-DRA promoter activity in the Rb-defective cells. Rb expression led to phosphorylation of Oct-1, thus relieving its repressive effect. Oct-1 has also been shown to repress interleukin 8 promoter activity. Consistent with reduced levels of Oct-1 DNA binding activity in the Rb-transformed cell lines, interleukin 8 expression is higher in these cell lines.

Functional association of TGF-beta receptor II with cyclin B.

Utilizing the cytoplasmic tail of Transforming Growth Factor Receptor Type II (TGFbeta RII) as bait in a yeast two hybrid system, we have identified human cyclin B2 as a direct physical partner of TGFbeta RII. Analysis of deletion mutants of glutathione-S-transferase (GST)-cyclin B2 mapped its binding domain for TGFbeta RII to the C-terminal and revealed a negative regulatory region immediately upstream of the cyclin box. Using recombinant proteins, Cdc2 was demonstrated to indirectly interact with TGFbeta RII via cyclin B2. This interaction was reproduced in THP-1 monocytic cells, where TGFbeta treatment markedly enhanced the ability of cyclin B2 and, correspondingly, Cdc2 from TGFbeta-treated THP-1 cells, to bind the GST-TGFbeta RII fusion protein. More importantly, TGFbeta RII co-precipitated with cyclin B2 in TGFbeta-treated THP-1 cells. TGFbeta treatment also caused threonine phosphorylation of Cdc2 in the TGFbeta RII-cyclin B2-Cdc2 complex in THP1 cells, in parallel with down regulation of Cdc2 function as measured by histone H1 kinase activity. Cyclin B1 had the same capacity to bind TGFbeta RII and mediate indirect Cdc2 binding. These results suggest an alternative mechanism that cell cycle arrest in the G1/S phase caused by TGFbeta may, in part, be due to inactivation of cyclin B/Cdc2 kinase, which is needed for entry into the G2/M phase.

Intact autocrine activation and cytokine production by PMNs from injured adults with elevated Candida antigen titres.

Injured patients with Candida antigen titres have increased mortality due to sepsis. Polymorphonuclear leucocytes (PMNs) from injured patients with elevated Candida antigen titres demonstrate impaired function against Candida albicans growth when compared with PMNs from injury matched controls. To determine if PMN dysfunction is global, PMNs from patients with positive Candida antigen titres were evaluated for their ability to activate the anticandidal function of normal PMNs (autocrine activation) and to produce tumour necrosis factor (TNF) and interleukin 8 (IL8), known activators of PMN anticandidal function, this study demonstrates that the PMN dysfunction is not global, as PMN cytokine production and autocrine activation remain intact.

Control of lytic function by mitogen-activated protein kinase/extracellular regulatory kinase 2 (ERK2) in a human natural killer cell line: identification of perforin and granzyme B mobilization by functional ERK2.

The signal pathways that control effector function in human natural killer (NK) cells are little known. In this study, we have identified the critical role of the mitogen-activated protein kinase (MAPK) pathway in NK lysis of tumor cells, and this pathway may involve the mobilization of granule components in NK cells upon interaction with sensitive tumor target cells. Evidence was provided by biological, biochemical, and gene transfection methods. NK cell binding to tumor cells for 5 min was sufficient to maximally activate MAPK/extracellular signal-regulatory kinase 2 (ERK2), demonstrated by its tyrosine phosphorylation and by its ability to function as an efficient kinase for myelin basic protein. MAPK activation was achieved in NK cells only after contact with NK-sensitive but not NK-resistant target cells. In immunocytochemical studies, cytoplasmic perforin and granzyme B were both maximally redirected towards the tumor contact zone within 5 min of NK cell contact with tumor cells. A specific MAPK pathway inhibitor, PD098059, could block not only MAPK activation but also redistribution of perforin/granzyme B in NK cells, which occur upon target ligation. PD098059 also interfered with NK lysis of tumor cells in a 5-h 51Cr-release assay, but had no ability to block NK cell proliferation. Transient transfection studies with wild-type and dominant-negative MAPK/ERK2 genes confirmed the importance of MAPK in NK cell lysis. These results document a pivotal role of MAPK in NK effector function, possibly by its control of movement of lytic granules, and clearly define MAPK involvement in a functional pathway unlinked to cell growth or differentiation.

Candida antigen titre dilution and death after injury.

BACKGROUND: Candida infections affect outcome after injury. Candida antigen titres were used to detect these infections early. This study was undertaken to correlate Candida antigen titre dilution with conventional injury scoring and outcome after severe injury. METHODS: Candida antigen titres were determined by agglutination when clinically apparent source(s) of Candida were noted, when Candida was grown in culture of body fluids, or when unexplained clinical deterioration occurred. The findings were compared with the Injury Severity Score (ISS). RESULTS: Seventy-five seriously injured adults (median ISS 25 (range 18-50)) developed raised Candida antigen titres. Multivariate analysis showed that age and Candida antigen titre correlated significantly with mortality, but not with each other. Culture evidence of Candida, or lack thereof, did not correlate with Candida antigen titre or mortality. Sixteen of 75 patients died, 14 from bacterial sepsis and none from Candida infection. CONCLUSION: In seriously injured adults, the mortality rate is related to raised Candida antigen titres. The association between Candida antigen titre and mortality, although real, remains unexplained.

CD40-CD40L interactions provide "third-party" costimulation for T cell response against B7-1-transfected human breast tumor cells.

In this study we provide evidence that a human breast carcinoma cell line, MDA-MB-231 (MDA), can be made immunogenic following B7 transfection and that full T cell activation is obtained through cooperation of T-B lymphocytes via CD40-CD40L interactions. Tumor cells transfected with either B7 gene (MDAB7), neomycin-resistant gene only (MDAneo), or untransfected (MDA) were used in an allogeneic mixed lymphocyte tumor culture (MLTC) to investigate their ability to stimulate T cell proliferation and generate cytotoxic T lymphocytes (CTL). MDAB7 induced moderate T cell proliferation while MDAneo or MDA did not. Substantial T cell proliferation and de novo generation of cytolytic T cells was obtained only in response to MDAB7 when B cells were present during the MLTC. CD8+-purified T + B cells proliferated to a greater extent than whole T cell populations + B or CD4+ + B in response to MDAB7. Addition of alpha-B7-1 or alpha-CD40 in the MLTC inhibited T cell proliferation by 65 and 40%, respectively, whereas T cell proliferation and generation of CTL was completely abrogated when MLTC was performed in the presence of both antibodies. These data suggest that the engagement of CD40L on T cells with CD40 on B cells provides a costimulatory signal which, in synergism with TCR-dependent MDAB7-T cell recognition (signal 1) and B7/CD28 interactions (signal 2), leads to full T cell activation.

Critical role of Lyn kinase in inhibition of neutrophil apoptosis by granulocyte-macrophage colony-stimulating factor.

The signal pathway for control of apoptosis in human neutrophils is currently unknown. In this study, we provide the first evidence that a Src family tyrosine kinase, Lyn, plays a key role in inhibition polymorphonuclear (PMN) cell death. Several nuclear proteins associated with apoptosis, i.e., p53, cdc2, and Rb, were absent from PMN. Bcl-2, known to inhibit apoptosis, was also not expressed. Programmed cell death that rapidly occurred in PMN could be arrested by granulocyte-macrophage CSF (GM-CSF), but this activation did not induce p53, cdc2, retinoblastoma, or Bcl-2 expression. Instead, GM-CSF produced a rapid activation of Lyn and Hck, but not Fgr, tyrosine phosphorylation within 1 min. Co-immunoprecipitation studies indicated that only Lyn, but not Hck, was physically coupled to GM-CSF receptor. By histologic assessment and evaluation of DNA fragmentation, only antisense Lyn, but not antisense Hck or antisense Fgr, could reverse the cell survival advantage provided by GM-CSF. Therefore, the physical coupling of Lyn to GM-CSF receptor and its early activation are required for inhibition or delay of apoptosis in PMN.

Identification of an amphotericin B resistant strain of Candida albicans using a rapid 3H-glucose incorporation microassay.

Using a 3H-glucose incorporation assay, antifungal sensitivity testing undertaken on an isolate of Candida albicans cultured from the blood of a bone marrow transplant patient documented resistance to amphotericin B but sensitivity to fluconazole and itraconazole. Information obtained from in vitro antifungal sensitivity testing can be used to direct in vivo antifungal therapy. Widespread application of standardized in vitro antifungal sensitivity testing is needed.

Injection of complementary DNA encoding interleukin-12 inhibits tumor establishment at a distant site in a murine renal carcinoma model.

Interleukin (IL-12) protein has been shown to elicit diverse immunological responses and potent antitumor activity. We demonstrate here that intradermal injection of IL-12 cDNA induces systemic biological effects characteristic of the cytokine in vivo. Intradermal injection of IL-12 cDNA resulted in local expression of IL-12 mRNA, which correlated with a 10-fold increase in natural killer activity and a 3-4-fold increase in anti-CD3-induced IFN-gamma production in cultured splenocytes. Furthermore, when challenged with Renca tumor cells at a distant site, the day of tumor emergence was significantly delayed, and tumor growth was reduced in mice that received IL-12 cDNA, compared to mice given injections of plasmid vector alone. A number of the mice receiving IL-12 cDNA injections remained tumor free months after tumor challenge. In contrast to mice receiving recombinant IL-12 protein, no splenomegaly was detected when natural killer activity was significantly induced in mice receiving injections of IL-12 cDNA. Because purified plasmid DNA is more economical to prepare and has a longer shelf-life than recombinant proteins, and intradermal administration of cDNA encoding IL-12 did not cause splenomegaly, our findings suggest that the in vivo injection of cDNA encoding IL-12 may be a less toxic and more cost-effective alternative to IL-12 protein therapy in some clinical or experimental therapeutic applications.

Interleukins 2 and 12 activate natural killer cytolytic responses of peripheral blood mononuclear cells from patients with head and neck squamous cell carcinoma.

OBJECTIVES: To examine the capacity of interleukin-2 (IL-2) and interleukin-12 (IL-12) to modulate the cytolytic activity of peripheral blood mononuclear cells against squamous cell carcinoma, and to determine whether peripheral blood mononuclear cells from healthy donors respond differently to IL-12 than do peripheral blood mononuclear cells from patients with head and neck squamous cell carcinoma. DESIGN: Case-comparison study of a consecutive sample of patients with head and neck squamous cell carcinoma who were scheduled to undergo surgical excision. PARTICIPANTS: The study included 10 patients with stage III or IV carcinoma matched with 10 volunteer blood donors. INTERVENTION: Isolated peripheral blood mononuclear cells from patients and volunteers were treated with IL-2, 10 U/mL and 100 U/mL; IL-12, 1 U/mL or 10 U/mL; or a combination of IL-2 and IL-12. RESULTS: The combination of IL-2 and IL-12 consistently produced the greatest activation cytolysis than either cytokine alone at all concentrations tested. This increased activity against a squamous cell carcinoma cell line was seen in lymphocytes from volunteers and patients. CONCLUSION: Our findings suggest a new treatment regimen for the patient with head and neck cancer that uses immunomodulation with a combination of cytokines.