Clonal cooperation through soluble metabolite exchange facilitates metastatic outgrowth by modulating Allee effect.

Cancers feature substantial intratumoral heterogeneity of genetic and phenotypically distinct lineages. Although interactions between coexisting lineages are emerging as a potential contributor to tumor evolution, the extent and nature of these interactions remain largely unknown. We postulated that tumors develop ecological interactions that sustain diversity and facilitate metastasis. Using a combination of fluorescent barcoding, mathematical modeling, metabolic analysis, and in vivo models, we show that the Allee effect, i.e., growth dependency on population size, is a feature of tumor lineages and that cooperative ecological interactions between lineages alleviate the Allee barriers to growth in a model of triple-negative breast cancer. Soluble metabolite exchange formed the basis for these cooperative interactions and catalyzed the establishment of a polyclonal community that displayed enhanced metastatic dissemination and outgrowth in xenograft models. Our results highlight interclonal metabolite exchange as a key modulator of tumor ecology and a contributing factor to overcoming Allee effect-associated growth barriers to metastasis.

Short-Term Exposure to Benzo(a)Pyrene Causes Disruption of GnRH Network in Zebrafish Embryos.

Benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon, is considered a common endocrine disrupting chemical (EDC) with mutagenic and carcinogenic effects. In this work, we evaluated the effects of BaP on the hypothalamo-pituitary-gonadal axis (HPG) of zebrafish embryos. The embryos were treated with 5 and 50 nM BaP from 2.5 to 72 hours post-fertilization (hpf) and obtained data were compared with those from controls. We followed the entire development of gonadotropin releasing hormone (GnRH3) neurons that start to proliferate from the olfactory region at 36 hpf, migrate at 48 hpf and then reach the pre-optic area and the hypothalamus at 72 hpf. Interestingly, we observed a compromised neuronal architecture of the GnRH3 network after the administration of 5 and 50 nM BaP. Given the toxicity of this compound, we evaluated the expression of genes involved in antioxidant activity, oxidative DNA damage and apoptosis and we found an upregulation of these pathways. Consequently, we performed a TUNEL assay and we confirmed an increment of cell death in brain of embryos treated with BaP. In conclusion our data reveal that short-term exposure of zebrafish embryos to BaP affects GnRH3 development likely through a neurotoxic mechanism.

HDAC6 inhibition decreases leukemic stem cell expansion driven by Hedgehog hyperactivation by restoring primary ciliogenesis.

Aberrant activation of the Hh pathway promotes cell proliferation and multi-drug resistance (MDR) in several cancers, including Acute Myeloid Leukemia (AML). Notably, only one Hh inhibitor, glasdegib, has been approved for AML treatment, and most patients eventually relapse, highlighting the urgent need to discover new therapeutic targets. Hh signal is transduced through the membrane of the primary cilium, a structure expressed by non-proliferating mammalian cells, whose stabilization depends on the activity of HDAC6. Here we describe a positive correlation between Hh, HDAC6, and MDR genes in a cohort of adult AML patients, human leukemic cell lines, and a zebrafish model of Hh overexpression. The hyper-activation of Hh or HDAC6 in zebrafish drove the increased proliferation of hematopoietic stem and progenitor cells (HSPCs). Interestingly, this phenotype was rescued by inhibition of HDAC6 but not of Hh. Also, in human leukemic cell lines, a reduction in vitality was obtained through HDAC6, but not Hh inhibition. Our data showed the presence of a cross-talk between Hh and HDAC6 mediated by stabilization of the primary cilium, which we detect for the first time in zebrafish HSPCs. Inhibition of HDAC6 activity alone or in combination therapy with the chemotherapeutic agent cytarabine, efficiently rescued the hematopoietic phenotype. Our results open the possibility to introduce HDAC6 as therapeutic target to reduce proliferation of leukemic blasts in AML patients.

Heme catabolism by tumor-associated macrophages controls metastasis formation.

Although the pathological significance of tumor-associated macrophage (TAM) heterogeneity is still poorly understood, TAM reprogramming is viewed as a promising anticancer therapy. Here we show that a distinct subset of TAMs (F4/80hiCD115hiC3aRhiCD88hi), endowed with high rates of heme catabolism by the stress-responsive enzyme heme oxygenase-1 (HO-1), plays a critical role in shaping a prometastatic tumor microenvironment favoring immunosuppression, angiogenesis and epithelial-to-mesenchymal transition. This population originates from F4/80+HO-1+ bone marrow (BM) precursors, accumulates in the blood of tumor bearers and preferentially localizes at the invasive margin through a mechanism dependent on the activation of Nrf2 and coordinated by the NF-κB1-CSF1R-C3aR axis. Inhibition of F4/80+HO-1+ TAM recruitment or myeloid-specific deletion of HO-1 blocks metastasis formation and improves anticancer immunotherapy. Relative expression of HO-1 in peripheral monocyte subsets, as well as in tumor lesions, discriminates survival among metastatic melanoma patients. Overall, these results identify a distinct cancer-induced HO-1+ myeloid subgroup as a new antimetastatic target and prognostic blood marker.

CD4 T Cell-Dependent Rejection of Beta-2 Microglobulin Null Mismatch Repair-Deficient Tumors.

Inactivation of beta-2 microglobulin (B2M) is considered a determinant of resistance to immune checkpoint inhibitors (ICPi) in melanoma and lung cancers. In contrast, B2M loss does not appear to affect response to ICPis in mismatch repair-deficient (MMRd) colorectal tumors where biallelic inactivation of B2M is frequently observed. We inactivated B2m in multiple murine MMRd cancer models. Although MMRd cells would not readily grow in immunocompetent mice, MMRd B2m null cells were tumorigenic and regressed when treated with anti-PD-1 and anti-CTLA4. The efficacy of ICPis against MMRd B2m null tumors did not require CD8+ T cells but relied on the presence of CD4+ T cells. Human tumors expressing low levels of B2M display increased intratumoral CD4+ T cells. We conclude that B2M inactivation does not blunt the efficacy of ICPi in MMRd tumors, and we identify a unique role for CD4+ T cells in tumor rejection. SIGNIFICANCE: B2M alterations, which impair antigen presentation, occur frequently in microsatellite-unstable colorectal cancers. Although in melanoma and lung cancers B2M loss is a mechanism of resistance to immune checkpoint blockade, we show that MMRd tumors respond to ICPis through CD4+ T-cell activation.This article is highlighted in the In This Issue feature, p. 1601.

MITO-Luc/GFP zebrafish model to assess spatial and temporal evolution of cell proliferation in vivo.

We developed a novel reporter transgenic zebrafish model called MITO-Luc/GFP zebrafish in which GFP and luciferase expression are under the control of the master regulator of proliferation NF-Y. In MITO-Luc/GFP zebrafish it is possible to visualize cell proliferation in vivo by fluorescence and bioluminescence. In this animal model, GFP and luciferase expression occur in early living embryos, becoming tissue specific in juvenile and adult zebrafish. By in vitro and ex vivo experiments we demonstrate that luciferase activity in adult animals occurs in intestine, kidney and gonads, where detectable proliferating cells are located. Further, by time lapse experiments in live embryos, we observed a wave of GFP positive cells following fin clip. In adult zebrafish, in addition to a bright bioluminescence signal on the regenerating tail, an early unexpected signal coming from the kidney occurs indicating not only a fin cell proliferation, but also a systemic response to tissue damage. Finally, we observed that luciferase activity was inhibited by anti-proliferative interventions, i.e. 5FU, cell cycle inhibitors and X-Rays. In conclusion, MITO-Luc/GFP zebrafish is a novel animal model that may be crucial to assess the spatial and temporal evolution of cell proliferation in vivo.

HDAC8: A Promising Therapeutic Target for Acute Myeloid Leukemia.

Histone deacetylase 8 (HDAC8), a class I HDAC that modifies non-histone proteins such as p53, is highly expressed in different hematological neoplasms including a subtype of acute myeloid leukemia (AML) bearing inversion of chromosome 16 [inv(16)]. To investigate HDAC8 contribution to hematopoietic stem cell maintenance and myeloid leukemic transformation, we generated a zebrafish model with Hdac8 overexpression and observed an increase in hematopoietic stem/progenitor cells, a phenotype that could be reverted using a specific HDAC8 inhibitor, PCI-34051 (PCI). In addition, we demonstrated that AML cell lines respond differently to PCI treatment: HDAC8 inhibition elicits cytotoxic effect with cell cycle arrest followed by apoptosis in THP-1 cells, and cytostatic effect in HL60 cells that lack p53. A combination of cytarabine, a standard anti-AML chemotherapeutic, with PCI resulted in a synergistic effect in all the cell lines tested. We, then, searched for a mechanism behind cell cycle arrest caused by HDAC8 inhibition in the absence of functional p53 and demonstrated an involvement of the canonical WNT signaling in zebrafish and in cell lines. Together, we provide the evidence for the role of HDAC8 in hematopoietic stem cell differentiation in zebrafish and AML cell lines, suggesting HDAC8 inhibition as a therapeutic target in hematological malignancies. Accordingly, we demonstrated the utility of a highly specific HDAC8 inhibition as a therapeutic strategy in combination with standard chemotherapy.

Tumor-Derived Prostaglandin E2 Promotes p50 NF-κB-Dependent Differentiation of Monocytic MDSCs.

Myeloid-derived suppressor cells (MDSC) include immature monocytic (M-MDSC) and granulocytic (PMN-MDSC) cells that share the ability to suppress adaptive immunity and to hinder the effectiveness of anticancer treatments. Of note, in response to IFNγ, M-MDSCs release the tumor-promoting and immunosuppressive molecule nitric oxide (NO), whereas macrophages largely express antitumor properties. Investigating these opposing activities, we found that tumor-derived prostaglandin E2 (PGE2) induces nuclear accumulation of p50 NF-κB in M-MDSCs, diverting their response to IFNγ toward NO-mediated immunosuppression and reducing TNFα expression. At the genome level, p50 NF-κB promoted binding of STAT1 to regulatory regions of selected IFNγ-dependent genes, including inducible nitric oxide synthase (Nos2). In agreement, ablation of p50 as well as pharmacologic inhibition of either the PGE2 receptor EP2 or NO production reprogrammed M-MDSCs toward a NOS2low/TNFαhigh phenotype, restoring the in vivo antitumor activity of IFNγ. Our results indicate that inhibition of the PGE2/p50/NO axis prevents MDSC-suppressive functions and restores the efficacy of anticancer immunotherapy. SIGNIFICANCE: Tumor-derived PGE2-mediated induction of nuclear p50 NF-κB epigenetically reprograms the response of monocytic cells to IFNγ toward an immunosuppressive phenotype, thus retrieving the anticancer properties of IFNγ. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/13/2874/F1.large.jpg.

RORC1 Regulates Tumor-Promoting "Emergency" Granulo-Monocytopoiesis.

Cancer-driven granulo-monocytopoiesis stimulates expansion of tumor promoting myeloid populations, mostly myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). We identified subsets of MDSCs and TAMs based on the expression of retinoic-acid-related orphan receptor (RORC1/RORγ) in human and mouse tumor bearers. RORC1 orchestrates myelopoiesis by suppressing negative (Socs3 and Bcl3) and promoting positive (C/EBPß) regulators of granulopoiesis, as well as the key transcriptional mediators of myeloid progenitor commitment and differentiation to the monocytic/macrophage lineage (IRF8 and PU.1). RORC1 supported tumor-promoting innate immunity by protecting MDSCs from apoptosis, mediating TAM differentiation and M2 polarization, and limiting tumor infiltration by mature neutrophils. Accordingly, ablation of RORC1 in the hematopoietic compartment prevented cancer-driven myelopoiesis, resulting in inhibition of tumor growth and metastasis.

The p50 subunit of NF-κB orchestrates dendritic cell lifespan and activation of adaptive immunity.

Dendritic cells play a central role in keeping the balance between immunity and immune tolerance. A key factor in this equilibrium is the lifespan of DC, as its reduction restrains antigen availability leading to termination of immune responses. Here we show that lipopolysaccharide-driven DC maturation is paralleled by increased nuclear levels of p50 NF-κB, an event associated with DC apoptosis. Lack of p50 in murine DC promoted increased lifespan, enhanced level of maturation associated with increased expression of the proinflammatory cytokines IL-1, IL-18 and IFN-ß, enhanced capacity of activating and expanding CD4(+) and CD8(+) T cells in vivo and decreased ability to induce differentiation of FoxP3(+) regulatory T cells. In agreement, vaccination of melanoma-bearing mice with antigen-pulsed LPS-treated p50(-/-) BM-DC boosted antitumor immunity and inhibition of tumor growth. We propose that nuclear accumulation of the p50 NF-κB subunit in DC, as occurring during lipopolysaccharide-driven maturation, is a homeostatic mechanism tuning the balance between uncontrolled activation of adaptive immunity and immune tolerance.

Origin and Functions of Tumor-Associated Myeloid Cells (TAMCs).

The construction of an inflammatory microenvironment provides the fuel for cancer development and progression. Hence, solid tumors promote the expansion and the recruitment of leukocyte populations, among which tumor-associated myeloid cells (TAMCs) represent a paradigm for cancer-promoting inflammation. TAMCs group heterogeneous phagocytic populations stemming from a common myeloid progenitor (CMP), that orchestrate various aspects of cancer, including: diversion and skewing of adaptive responses; immunosuppression; cell growth; angiogenesis; matrix deposition and remodelling; construction of a metastatic niche and actual metastasis. Several evidence indicate that TAMCs show plasticity and/or functional heterogeneity, suggesting that tumour-derived factors promote their functional "reprogramming" towards protumoral activities. While recent studies have attempted to address the role of microenvironment signals, the interplay between cancer cells, innate and adaptive immunity is now emerging as a crucial step of the TAMCs reprogramming. Here we discuss the evidence for the differentiation of TAMCs during the course of tumor progression and the molecular mechanisms that regulate such event.

Enhanced interferon regulatory factor 3 binding to the interleukin-23p19 promoter correlates with enhanced interleukin-23 expression in systemic lupus erythematosus.

OBJECTIVE: To examine the role of interferon regulatory factor 3 (IRF-3) in the regulation of interleukin-23 (IL-23) production in patients with systemic lupus erythematosus (SLE). METHODS: Bone marrow-derived macrophages were isolated from both wild-type and IRF3(-/-) C57BL/6 mice. These cells were stimulated with the Toll-like receptor 3 (TLR-3) agonist poly(I-C), and IL-23p19 cytokine levels were analyzed by enzyme-linked immunosorbent assay. IRF-3 binding to the IL-23p19 gene promoter region in monocytes from patients with SLE and healthy control subjects was analyzed by chromatin immunoprecipitation (ChIP) assay. Luciferase reporter gene assays were performed to identify key drivers of IL-23p19 promoter activity. TANK-binding kinase 1 (TBK-1) protein levels were determined by Western blotting. RESULTS: ChIP assays demonstrated that IRF-3 was stably bound to the human IL-23p19 promoter in monocytes; this association increased following TLR-3 stimulation. Patients with SLE demonstrated increased levels of IRF-3 bound to the IL-23p19 promoter compared with control subjects, which correlated with enhanced IL-23p19 production in monocytes from patients with SLE. Investigations of the TLR-3-driven responses in monocytes from patients with SLE revealed that TBK-1, which is critical for regulating IRF-3 activity, was hyperactivated in both resting and TLR-3-stimulated cells. CONCLUSION: Our results demonstrate for the first time that patients with SLE display enhanced IL-23p19 expression as a result of hyperactivation of TBK-1, resulting in increased binding of IRF-3 to the promoter. These findings provide novel insights into the molecular pathogenesis of SLE and the potential role for TLR-3 in driving this response.

Macrophages in cancer and infectious diseases: the 'good' and the 'bad'.

Macrophages are crucial orchestrators of host defence and tissue homeostasis. Macrophages are heterogeneous and plastic cells that in response to different microenvironmental signals can mount a broad spectrum of different programs of polarized activation. In different pathological contexts including cancer and infectious diseases, macrophages diversity and plasticity may act as a double-edged sword. The elucidation of the molecular mechanisms underlying macrophages recruitment and functional activation allows the identification of valuable targets for the development of innovative therapeutic approaches.

Cellular and molecular pathways linking inflammation and cancer.

Several experimental and epidemiological evidence indicate that, irrespective of the trigger for the development (chronic infection/inflammation or genetic alteration), a "smouldering" inflammation is associated with the most of, if not all, tumours and supports their progression. Several evidence have highlighted that tumours promote a constant influx of myelomonocytic cells that express inflammatory mediators supporting pro-tumoral functions. Myelomonocytic cells are key orchestrators of cancer-related inflammation associated with proliferation and survival of malignant cells, subversion of adaptive immune response, angiogenesis, stroma remodelling and metastasis formation. Although the connection between inflammation and cancer is unequivocal the mechanistic basis of such association are largely unknown. Recent advances in the understanding of the cellular and molecular pathways involved in cancer-related inflammation as well as their potential relevance as diagnostic, prognostic and therapeutic targets are herein discussed.

The histone H3 lysine 27-specific demethylase Jmjd3 is required for neural commitment.

Patterns of methylation at lysine 4 and 27 of histone H3 have been associated with states of gene activation and repression that are developmentally regulated and are thought to underlie the establishment of lineage specific gene expression programs. Recent studies have provided fundamental insight into the problem of lineage specification by comparing global changes in chromatin and transcription between ES and neural stem (NS) cells, points respectively of departure and arrival for neural commitment. With these maps of the differentiated state in place, a central task is now to unravel the chromatin dynamics that enables these differentiation transitions. In particular, the observation that lineage-specific genes repressed in ES cells by Polycomb-mediated H3-K27 trimethylation (H3-K27me3) are demethylated and derepressed in differentiated cells posited the existence of a specific H3-K27 demethylase.In order to gain insight into the epigenetic transitions that enable lineage specification, we investigated the early stages of neural commitment using as model system the monolayer differentiation of mouse ES cells into neural stem (NS) cells. Starting from a comprehensive profiling of JmjC-domain genes, we report here that Jmjd3, recently identified as a H3-K27me3 specific demethylase, controls the expression of key regulators and markers of neurogenesis and is required for commitment to the neural lineage.Our results demonstrate the relevance of an enzymatic activity that antagonizes Polycomb regulation and highlight different modalities through which the dynamics of H3-K27me3 is related to transcriptional output. By showing that the H3-K27 demethylase Jmjd3 is required for commitment to the neural lineage and that it resolves the bivalent domain at the Nestin promoter, our work confirms the functional relevance of bivalent domain resolution that had been posited on the basis of the genome-wide correlation between their controlled resolution and differentiation. In addition, our data indicate that the regulation of H3-K27me3 is highly gene- and context- specific, suggesting that the interplay of methyltransferases and demethylases enables the fine-tuning more than the on/off alternation of methylation states.

Macrophage polarization in tumour progression.

Macrophages are a fundamental part of the innate defense mechanisms, which can promote specific immunity by inducing T cell recruitment and activation. Despite this, their presence within the tumour microenvironment has been associated with enhanced tumour progression and shown to promote cancer cell growth and spread, angiogenesis and immunosuppression. This paradoxical role of macrophages in cancer finds an explanation in their functional plasticity, that may result in the polarized expression of either pro- or anti-tumoural functions. Key players in the setting of their phenotype are the microenvironmental signals to which macrophages are exposed, which selectively tune their functions within a functional spectrum encompassing the M1 and M2 extremes. Here, we discuss recent findings suggesting that targeting tumour-associated macrophages (TAMs) polarization may represent a novel therapeutic strategy against cancer.

The histone H3 lysine-27 demethylase Jmjd3 links inflammation to inhibition of polycomb-mediated gene silencing.

Epigenetic chromatin marks restrict the ability of differentiated cells to change gene expression programs in response to environmental cues and to transdifferentiate. Polycomb group (PcG) proteins mediate gene silencing and repress transdifferentiation in a manner dependent on histone H3 lysine 27 trimethylation (H3K27me3). However, macrophages migrated into inflamed tissues can transdifferentiate, but it is unknown whether inflammation alters PcG-dependent silencing. Here we show that the JmjC-domain protein Jmjd3 is a H3K27me demethylase expressed in macrophages in response to bacterial products and inflammatory cytokines. Jmjd3 binds PcG target genes and regulates their H3K27me3 levels and transcriptional activity. The discovery of an inducible enzyme that erases a histone mark controlling differentiation and cell identity provides a link between inflammation and reprogramming of the epigenome, which could be the basis for macrophage plasticity and might explain the differentiation abnormalities in chronic inflammation.