CXCR4/CXCL12 Activities in the Tumor Microenvironment and Implications for Tumor Immunotherapy.

CXCR4 is a G-Protein coupled receptor that is expressed nearly ubiquitously and is known to control cell migration via its interaction with CXCL12, the most ancient chemokine. The functions of CXCR4/CXCL12 extend beyond cell migration and involve the recognition and disposal of unhealthy or tumor cells. The CXCR4/CXCL12 axis plays a relevant role in shaping the tumor microenvironment (TME), mainly towards dampening immune responses. Notably, CXCR4/CXCL12 cross-signal via the T and B cell receptors (TCR and BCR) and co-internalize with CD47, promoting tumor cell phagocytosis by macrophages in an anti-tumor immune process called ImmunoGenic Surrender (IGS). These specific activities in shaping the immune response might be exploited to improve current immunotherapies.

Use of an antagonist of HMGB1 in mice affected by malignant mesothelioma: a preliminary ultrasound and optical imaging study.

BACKGROUND: Malignant mesothelioma (MM) is an aggressive tumor, with a poor prognosis, usually unresectable due to late diagnosis, mainly treated with chemotherapy. BoxA, a truncated form of "high mobility group box 1" (HMGB1), acting as an HMGB1 antagonist, might exert a defensive action against MM. We investigated the potential of BoxA for MM treatment using experimental 40-MHz ultrasound and optical imaging (OI) in a murine model. METHODS: Murine MM cells infected with a lentiviral vector expressing the luciferase gene were injected into the peritoneum of 14 BALB/c mice (7 × 104 AB1-B/c-LUC cells). These mice were randomized to treatment with BoxA (n = 7) or phosphate-buffered saline (controls, n = 7). The experiment was repeated with 40 mice divided into two groups (n = 20 + 20) and treated as above to confirm the result and achieve greater statistical power. Tumor presence was investigated by experimental ultrasound and OI; suspected peritoneal masses underwent histopathology and immunohistochemistry examination. RESULTS: In the first experiment, none of the 7 controls survived beyond day 27, whereas 4/7 BoxA-treated mice (57.1%) survived up to day 70. In the second experiment, 6/20 controls (30.0%) and 16/20 BoxA-treated mice (80.0%) were still alive at day 34 (p = 0.004). In both experiments, histology confirmed the malignant nature of masses detected using experimental ultrasound and OI. CONCLUSION: In our preclinical experience on a murine model, BoxA seems to exert a protective role toward MM. Both experimental ultrasound and OI proved to be reliable techniques for detecting MM peritoneal masses.

CXCR4 engagement triggers CD47 internalization and antitumor immunization in a mouse model of mesothelioma.

Boosting antitumor immunity has emerged as a powerful strategy in cancer treatment. While releasing T-cell brakes has received most attention, tumor recognition by T cells is a pre-requisite. Radiotherapy and certain cytotoxic drugs induce the release of damage-associated molecular patterns, which promote tumor antigen cross-presentation and T-cell priming. Antibodies against the "do not eat me" signal CD47 cause macrophage phagocytosis of live tumor cells and drive the emergence of antitumor T cells. Here we show that CXCR4 activation, so far associated only with tumor progression and metastasis, also flags tumor cells to immune recognition. Both CXCL12, the natural CXCR4 ligand, and BoxA, a fragment of HMGB1, promote the release of DAMPs and the internalization of CD47, leading to protective antitumor immunity. We designate as Immunogenic Surrender the process by which CXCR4 turns in tumor cells to macrophages, thereby subjecting a rapidly growing tissue to immunological scrutiny. Importantly, while CXCL12 promotes tumor cell proliferation, BoxA reduces it, and might be exploited for the treatment of malignant mesothelioma and a variety of other tumors.

High-mobility group box 1 protein orchestrates responses to tissue damage via inflammation, innate and adaptive immunity, and tissue repair.

A single protein, HMGB1, directs the triggering of inflammation, innate and adaptive immune responses, and tissue healing after damage. HMGB1 is the best characterized damage-associated molecular pattern (DAMP), proteins that are normally inside the cell but are released after cell death, and allow the immune system to distinguish between antigens that are dangerous or not. Notably, cells undergoing severe stress actively secrete HMGB1 via a dedicated secretion pathway: HMGB1 is relocated from the nucleus to the cytoplasm and then to secretory lysosomes or directly to the extracellular space. Extracellular HMGB1 (either released or secreted) triggers inflammation and adaptive immunological responses by switching among multiple oxidation states, which direct the mutually exclusive choices of different binding partners and receptors. Immune cells are first recruited to the damaged tissue and then activated; thereafter, HMGB1 supports tissue repair and healing, by coordinating the switch of macrophages to a tissue-healing phenotype, activation and proliferation of stem cells, and neoangiogenesis. Inevitably, HMGB1 also orchestrates the support of stressed but illegitimate tissues: tumors. Concomitantly, HMGB1 enhances the immunogenicity of mutated proteins in the tumor (neoantigens), promoting anti-tumor responses and immunological memory. Tweaking the activities of HMGB1 in inflammation, immune responses and tissue repair could bring large rewards in the therapy of multiple medical conditions, including cancer.

Human malignant mesothelioma is recapitulated in immunocompetent BALB/c mice injected with murine AB cells.

Malignant Mesothelioma is a highly aggressive cancer, which is difficult to diagnose and treat. Here we describe the molecular, cellular and morphological characterization of a syngeneic system consisting of murine AB1, AB12 and AB22 mesothelioma cells injected in immunocompetent BALB/c mice, which allows the study of the interplay of tumor cells with the immune system. Murine mesothelioma cells, like human ones, respond to exogenous High Mobility Group Box 1 protein, a Damage-Associated Molecular Pattern that acts as a chemoattractant for leukocytes and as a proinflammatory mediator. The tumors derived from AB cells are morphologically and histologically similar to human MM tumors, and respond to treatments used for MM patients. Our system largely recapitulates human mesothelioma, and we advocate its use for the study of MM development and treatment.

Down syndrome fibroblasts and mouse Prep1-overexpressing cells display increased sensitivity to genotoxic stress.

PREP1 (PKNOX1) maps in the Down syndrome (DS) critical region of chromosome 21, is overexpressed in some DS tissues and might be involved in the DS phenotype. By using fibroblasts from DS patients and by overexpressing Prep1 in F9 teratocarcinoma and Prep1(i/i) MEF to single out the role of the protein, we report that excess Prep1 increases the sensitivity of cells to genotoxic stress and the extent of the apoptosis directly correlates with the level of Prep1. The apoptotic response of Prep1-overexpressing cells is mediated by the pro-apoptotic p53 protein that we show is a direct target of Prep1, as its depletion reverts the apoptotic phenotype. The induction of p53 overcomes the anti-apoptotic role of Bcl-X(L), previously shown to be also a Prep1 target, the levels of which are increased in Prep1-overexpressing cells as well. Our results provide a rationale for the involvement of PREP1 in the apoptotic phenotype of DS tissues and indicate that differences in Prep1 level can have drastic effects.

Poised transcription factories prime silent uPA gene prior to activation.

The position of genes in the interphase nucleus and their association with functional landmarks correlate with active and/or silent states of expression. Gene activation can induce chromatin looping from chromosome territories (CTs) and is thought to require de novo association with transcription factories. We identify two types of factory: "poised transcription factories," containing RNA polymerase II phosphorylated on Ser5, but not Ser2, residues, which differ from "active factories" associated with phosphorylation on both residues. Using the urokinase-type plasminogen activator (uPA) gene as a model system, we find that this inducible gene is predominantly associated with poised (S5p(+)S2p(-)) factories prior to activation and localized at the CT interior. Shortly after induction, the uPA locus is found associated with active (S5p(+)S2p(+)) factories and loops out from its CT. However, the levels of gene association with poised or active transcription factories, before and after activation, are independent of locus positioning relative to its CT. RNA-FISH analyses show that, after activation, the uPA gene is transcribed with the same frequency at each CT position. Unexpectedly, prior to activation, the uPA loci internal to the CT are seldom transcriptionally active, while the smaller number of uPA loci found outside their CT are transcribed as frequently as after induction. The association of inducible genes with poised transcription factories prior to activation is likely to contribute to the rapid and robust induction of gene expression in response to external stimuli, whereas gene positioning at the CT interior may be important to reinforce silencing mechanisms prior to induction.

Induction of HoxB transcription by retinoic acid requires actin polymerization.

We have analyzed the role of actin polymerization in retinoic acid (RA)-induced HoxB transcription, which is mediated by the HoxB regulator Prep1. RA induction of the HoxB genes can be prevented by the inhibition of actin polymerization. Importantly, inhibition of actin polymerization specifically affects the transcription of inducible Hox genes, but not that of their transcriptional regulators, the RARs, nor of constitutively expressed, nor of actively transcribed Hox genes. RA treatment induces the recruitment to the HoxB2 gene enhancer of a complex composed of "elongating" RNAPII, Prep1, beta-actin, and N-WASP as well as the accessory splicing components p54Nrb and PSF. We show that inhibition of actin polymerization prevents such recruitment. We conclude that inducible Hox genes are selectively sensitive to the inhibition of actin polymerization and that actin polymerization is required for the assembly of a transcription complex on the regulatory region of the Hox genes.

Prep1 directly regulates the intrinsic apoptotic pathway by controlling Bcl-XL levels.

The Prep1 homeodomain transcription factor is essential in embryonic development. Prep1 hypomorphic mutant mouse (Prep1(i/i)) embryos (embryonic day 9.5) display an increased terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling reaction compared to wild-type (WT) littermates. Prep1(i/i) mouse embryo fibroblasts (MEFs) show an increased basal level of annexin V binding activity, reduction of the mitochondrial-membrane potential, and increased caspase 9 and 3 activation, indicating increased apoptosis. Prep1(i/i) MEFs also respond faster than WT MEFs to genotoxic stress, indicating increased activation of the intrinsic apoptotic pathways. We did not observe an increase in p53 or an abnormal p53 response to apoptotic stimuli. However, hypomorphic MEFs have decreased endogenous levels of antiapoptotic Bcl-X(L) mRNA and protein, and Bcl-x overexpression rescues the defect of Prep1(i/i) MEFs. Using transient transfections and chromatin immunoprecipitation, we identified the Bcl-x promoter as a novel target of Prep1. Thus, Prep1 directly controls mitochondrial homeostasis (and the apoptotic potential) by modulating Bcl-x gene expression.

A transcription-dependent micrococcal nuclease-resistant fragment of the urokinase-type plasminogen activator promoter interacts with the enhancer.

We show the interaction between the enhancer and the minimal promoter of urokinase-type plasminogen activator gene during active transcription by coupling micrococcal nuclease digestion of cross-linked, sonicated chromatin, and chromatin immunoprecipitation. This approach allowed the precise identification of the interacting genomic fragments, one of which is resistant to micrococcal nuclease cleavage. The interacting fragments form a single transcriptional control unit, as indicated by their common protein content. Furthermore, we show that the enhancer-MP interaction persists during the early stages of transcription and is lost upon alpha-amanitin treatment, indicating the requirement for active transcription. Our results support a looping model of interaction between the enhancer and the MP of the urokinase-type plasminogen activator gene.

Urokinase-type plasminogen activator.

Urokinase-type plasminogen activator (uPA) is a serine protease involved in tissue remodeling and cell migration. At the gene level, the interplay between a complex enhancer, required for induced and basal transcription, and the minimal promoter finely tunes uPA expression. The active form of uPA is bound to its high affinity receptor on the cell surface, where specific inhibitors modulate its enzymatic activity. Such inhibitors also regulate the cell surface levels of uPA by triggering the internalization of the uPA-receptor-inhibitor complex. The role of uPA is not only linked to its action as an enzyme. In fact, the mere binding of uPA on the cell surface also brings about two events that broaden the spectrum of its biological functions: (1) a conformational change of the receptor, which, in turn, affects its interaction with other proteins; (2) a signal transduction which modulates the expression of apoptosis-related genes. Besides its applications as a thrombolytic agent and as a prognostic marker for tumors, uPA may provide the basis for other therapies, as the structure of the receptor-binding domain of uPA has become a model for the design of anti-cancer molecules.

Nuclear myosin VI enhances RNA polymerase II-dependent transcription.

Myosin VI is the only myosin that moves toward the minus end of actin filaments, suggesting a unique biological function. Here, we show that myosin VI is present in the nucleus of mammalian cells where it colocalizes with newly transcribed mRNA and with RNA polymerase II (RNAPII) and is detected in the RNAPII complex. The colocalization and interaction of myosin VI with RNAPII require transcriptional activity. Chromatin immunoprecipitation (ChIP) demonstrates that myosin VI is recruited to the promoter and intragenic regions of active genes, encoding urokinase plasminogen activator (uPA), eukaryotic initiation factor 6 (p27/eIF6), and low-density lipoprotein receptor (LDLR), but not to noncoding, nonregulatory intergenic regions. Downregulation of myosin VI reduces steady-state mRNA levels of these genes in vivo, and antibodies to myosin VI reduce transcription in vitro. We suggest that myosin VI modulates RNAPII-dependent transcription of active genes, implicating the possibility of an actin-myosin based mechanism of transcription.

Pertussis toxin B-oligomer suppresses IL-6 induced HIV-1 and chemokine expression in chronically infected U1 cells via inhibition of activator protein 1.

Pertussis toxin B-oligomer (PTX-B) inhibits HIV replication in T lymphocytes and monocyte-derived macrophages by interfering with multiple steps of the HIV life cycle. PTX-B prevents CCR5-dependent (R5) virus entry in a noncompetitive manner, and it also exerts suppressive effects on both R5- and CXCR4-dependent HIV expression at a less-characterized postentry level. We demonstrate in this study that PTX-B profoundly inhibits HIV expression in chronically infected promonocytic U1 cells stimulated with several cytokines and, particularly, the IL-6-mediated effect, a cytokine that triggers viral production in these cells independently of NF-kappaB activation. From U1 cells we have subcloned a cell line, named U1-CR1, with increased responsiveness to IL-6. In these cells, PTX-B neither down-regulated the IL-6R nor prevented IL-6 induced signaling in terms of STAT3 phosphorylation and DNA binding. In contrast, PTX-B inhibited AP-1 binding to target DNA and modified its composition with a proportional increases in FosB, Fra2, and ATF2. PTX-B inhibited IL-6-induced HIV-1 long-terminal repeat-driven transcription from A, C, E, and F viral subtypes, which contain functional AP-1 binding sites, but failed to inhibit transcription from subtypes B and D LTR devoid of these sites. In addition, PTX-B inhibited the secretion of IL-6-induced, AP-1-dependent genes, including urokinase-type plasminogen activator, CXCL8/IL-8, and CCL2/monocyte chemotactic protein-1. Thus, PTX-B suppression of IL-6 induced expression of HIV and cellular genes in chronically infected promonocytic cells is strongly correlated to inhibition of AP-1.

MAPK and JNK transduction pathways can phosphorylate Sp1 to activate the uPA minimal promoter element and endogenous gene transcription.

Two upstream regions of the human urokinase (uPA) gene regulate its transcription: the minimal promoter (MP) and the enhancer element. The activity of the minimal promoter is essential for basal uPA transcription in prostate adenocarcinoma PC3 cells. Binding of a phosphorylated Sp1 transcription factor is, in turn, essential for the activity of the MP. Here we report that the Jun kinase (JNK) pathway is required for the basal activity of the MP and for the expression of the endogenous uPA gene in PC3 cells and for activated transcription in LNCaP cells. On the other hand, the p42/p44 mitogen-activated protein kinase (MAPK) pathway activates uPA gene expression through Sp1 phosphorylation in HeLa, LNCaP, and CCL39-derivative cells that do not typically express uPA in basal conditions. In HeLa cells the dominant-negative form of JNK interferes with the p42/p44 MAPK activation of the uPA-MP. The results suggest that the stress-activated protein kinase (SAPK)/JNK pathway plays an important role in the phosphorylation of Sp1, which, in turn, leads to basal or activated transcription from the uPA-MP element.

Binding of Sp1 to the proximal promoter links constitutive expression of the human uPA gene and invasive potential of PC3 cells.

Activated transcription of the urokinase-type plasminogen activator (uPA) gene depends on the enhancer, located approximately 2 kb from the start of transcription. The proximal promoter, driving basal transcription, contains a GC-/GA-rich sequence immediately upstream of the TATA box. We have investigated the role played by this element in the transcription of the uPA gene in HeLa and PC3 cells, which do not express or constitutively express the gene, respectively. This region binds either Sp1 or Sp3, as monomers or multimers, but not a combination of the 2 proteins. The more efficient binding of Sp1 to the proximal promoter in PC3 cells is correlated to its phosphorylation state. Polymerase chain reaction (PCR)-coupled, chromatin immunoprecipitation experiments with anti-Sp1 antibodies indeed show an enrichment of proximal promoter sequences in PC3 cells and support the observed difference in transcription levels from proximal promoter constructs in HeLa versus PC3 cells. Furthermore, overexpression of Sp1 increases transcription from the reporter construct in HeLa cells, whereas in PC3 cells, overexpression of Sp3 does not reduce transcription from the same construct, indicating that the Sp1/Sp3 balance cannot be shifted. We conclude that the GC-/GA-rich element of the uPA regulatory region is an independent functional element, regulated by Sp family proteins. Phosphorylation of Sp1 determines the presence in vivo and the functionality of this element in PC3 cells. Thus, the cellular context determines the relevance of the GC-/GA-rich region in uPA gene transcription, which contributes to constitutive gene expression, related, in turn, to the invasive phenotype.