MAPK Signaling Is Required for Generation of Tunneling Nanotube-Like Structures in Ovarian Cancer Cells.

Ovarian cancer (OC) cells survive in the peritoneal cavity in a complex microenvironment composed of diverse cell types. The interaction between tumor cells and non-malignant cells is crucial to the success of the metastatic process. Macrophages activate pro-metastatic signaling pathways in ovarian cancer cells (OCCs), induce tumor angiogenesis, and orchestrate a tumor suppressive immune response by releasing anti-inflammatory cytokines. Understanding the interaction between immune cells and tumor cells will enhance our ability to combat tumor growth and dissemination. When co-cultured with OCCs, macrophages induce projections consistent with tunneling nanotubes (TnTs) to form between OCCs. TnTs mediate transfer of material between cells, thus promoting invasiveness, angiogenesis, proliferation, and/or therapy resistance. Macrophage induction of OCC TnTs occurs through a soluble mediator as macrophage-conditioned media potently induced TnT formation in OCCs. Additionally, EGFR-induced TnT formation in OCCs through MAPK signaling may occur. In particular, inhibition of ERK and RSK prevented EGFR-induced TnTs. TnT formation in response to macrophage-conditioned media or EGFR signaling required MAPK signaling. Collectively, these studies suggest that inhibition of ERK/RSK activity may dampen macrophage-OCC communication and be a promising therapeutic strategy.

The miR-23a∼27a∼24-2 microRNA Cluster Promotes Inflammatory Polarization of Macrophages.

Macrophages are critical for regulating inflammatory responses. Environmental signals polarize macrophages to either a proinflammatory (M1) state or an anti-inflammatory (M2) state. We observed that the microRNA (miRNA) cluster mirn23a, coding for miRs-23a, -27a, and -24-2, regulates mouse macrophage polarization. Gene expression analysis of mirn23a-deficient myeloid progenitors revealed a decrease in TLR and IFN signaling. Mirn23a -/- bone marrow-derived macrophages (BMDMs) have an attenuated response to LPS, demonstrating an anti-inflammatory phenotype in mature cells. In vitro, mirn23a-/- BMDMs have decreased M1 responses and an enhanced M2 responses. Overexpression of mirn23a has the opposite effect, enhancing M1 and inhibiting M2 gene expression. Interestingly, expression of mirn23a miRNAs goes down with inflammatory stimulation and up with anti-inflammatory stimulation, suggesting that its regulation prevents locking macrophages into polarized states. M2 polarization of tumor-associated macrophages (TAMs) correlates with poor outcome for many tumors, so to determine if there was a functional consequence of mirn23a loss modulating immune cell polarization, we assayed syngeneic tumor growth in wild-type and mirn23a -/- mice. Consistent with the increased anti-inflammatory/immunosuppressive phenotype in vitro, mirn23a -/- mice inoculated with syngeneic tumor cells had worse outcomes compared with wild-type mice. Coinjecting tumor cells with mirn23a -/- BMDMs into wild-type mice phenocopied tumor growth in mirn23a -/- mice, supporting a critical role for mirn23a miRNAs in macrophage-mediated tumor immunity. Our data demonstrate that mirn23a regulates M1/M2 polarization and suggests that manipulation of mirn23a miRNA can be used to direct macrophage polarization to drive a desired immune response.

Brucella suppress STING expression via miR-24 to enhance infection.

Brucellosis, caused by a number of Brucella species, remains the most prevalent zoonotic disease worldwide. Brucella establish chronic infections within host macrophages despite triggering cytosolic innate immune sensors, including Stimulator of Interferon Genes (STING), which potentially limit infection. In this study, STING was required for control of chronic Brucella infection in vivo. However, early during infection, Brucella down-regulated STING mRNA and protein. Down-regulation occurred post-transcriptionally, required live bacteria, the Brucella type IV secretion system, and was independent of host IRE1-RNase activity. STING suppression occurred in MyD88-/- macrophages and was not induced by Toll-like receptor agonists or purified Brucella lipopolysaccharide (LPS). Rather, Brucella induced a STING-targeting microRNA, miR-24-2, in a type IV secretion system-dependent manner. Furthermore, STING downregulation was inhibited by miR-24 anti-miRs and in Mirn23a locus-deficient macrophages. Failure to suppress STING expression in Mirn23a-/- macrophages correlated with diminished Brucella replication, and was rescued by exogenous miR-24. Mirn23a-/- mice were also more resistant to splenic colonization one week post infection. Anti-miR-24 potently suppressed replication in wild type, but much less in STING-/- macrophages, suggesting most of the impact of miR-24 induction on replication occurred via STING suppression. In summary, Brucella sabotages cytosolic surveillance by miR-24-dependent suppression of STING expression; post-STING activation "damage control" via targeted STING destruction may enable establishment of chronic infection.

ARID3A and ARID3B induce stem promoting pathways in ovarian cancer cells.

ARID3A and ARID3B are paralogs from the AT-Rich interactive Domain (ARID) family. ARID3A and ARID3B associate to regulate genes in B-cells and cancer. We were the first to demonstrate that ARID3B regulates stem cell genes and promotes the cancer stem cell phenotype. Importantly, different knockout phenotypes in mice and distinct patterns of expression in adult animals suggests that ARID3A and ARID3B may have unique functions. In addition, high levels of ARID3B but not ARID3A induce cell death. Our goal was to express ARID3A, ARID3B, or both genes at a moderate level (as can be observed in cancer) and then identify ARID3 regulated genes. We transduced ovarian cancer cells with ARID3A-GFP, ARID3B-RFP, or both. RNA-sequencing was conducted. ARID3A and ARID3B regulated nearly identical sets of genes. Few genes (<5%) were uniquely regulated by ARID3A or ARID3B. ARID3A/B induced genes involved in cancer and stem cell processes including: Twist, MYCN, MMP2, GLI2, TIMP3, and WNT5B. We found that ARID3A and ARID3B also induced expression of each other, providing evidence of the cooperativity. While ARID3A and ARID3B likely have unique functions in distinct contexts, they are largely capable of regulating the same stem cell genes in cancer cells. This study provides a comprehensive list of genes and pathways regulated by ARID3A and ARID3B in ovarian cancer cells.

Mirc11 Disrupts Inflammatory but Not Cytotoxic Responses of NK Cells.

Natural killer (NK) cells generate proinflammatory cytokines that are required to contain infections and tumor growth. However, the posttranscriptional mechanisms that regulate NK cell functions are not fully understood. Here, we define the role of the microRNA cluster known as Mirc11 (which includes miRNA-23a, miRNA-24a, and miRNA-27a) in NK cell-mediated proinflammatory responses. Absence of Mirc11 did not alter the development or the antitumor cytotoxicity of NK cells. However, loss of Mirc11 reduced generation of proinflammatory factors in vitro and interferon-γ-dependent clearance of Listeria monocytogenes or B16F10 melanoma in vivo by NK cells. These functional changes resulted from Mirc11 silencing ubiquitin modifiers A20, Cbl-b, and Itch, allowing TRAF6-dependent activation of NF-κB and AP-1. Lack of Mirc11 caused increased translation of A20, Cbl-b, and Itch proteins, resulting in deubiquitylation of scaffolding K63 and addition of degradative K48 moieties on TRAF6. Collectively, our results describe a function of Mirc11 that regulates generation of proinflammatory cytokines from effector lymphocytes.

The mirn23a and mirn23b microrna clusters are necessary for proper hematopoietic progenitor cell production and differentiation.

Mice deficient for microRNA (miRNA) cluster mirn23a exhibit increased B lymphopoiesis at the expense of myelopoiesis, whereas hematopoietic stem and progenitor cell (HSPC) populations are unchanged. Mammals possess a paralogous mirn23b gene that can give rise to three mature miRNAs (miR-23b, miR-24-1, and miR-27b) that have identical seed/mRNA-targeting sequences to their mirn23a counterparts. To assess whether compound deletion of mirn23a and mirn23b exacerbates the hematopoietic phenotype observed in mirn23a-/- mice, we generated a compound mirn23a-/-mirn23bfl/fl:Mx1-Cre conditional knockout mouse and assayed hematopoietic development after excision of mirn23b. Loss of both genes in adult bone marrow further skewed HSPC differentiation toward B cells at the expense of myeloid cells, demonstrating a dosage-dependent effect on regulating cell differentiation. Strikingly, double-knockout (DKO) mice had decreased bone marrow cellularity with significantly decreased hematopoietic stem cell and HSPC populations, a phenotype not observed in mice deficient for mirn23a alone. Competitive transplantation assays showed decreased contribution of mirn23a-/-mirn23b-/- HSPCs to hematopoietic lineages at 6 and 12 weeks after transplantation. Defects in the proliferation of mirn23a-/-b-/- HSPCs was not observed; however, DKO cells were more apoptotic compared with both wild-type and mirn23a-/- cells. Together, our data show that complete loss of mirn23a/mirn23b miRNAs results in decreased blood production and affects lineage output in a concentration-dependent manner.

The miR-23a~27a~24-2 microRNA cluster buffers transcription and signaling pathways during hematopoiesis.

MicroRNA cluster mirn23a has previously been shown to promote myeloid development at the expense of lymphoid development in overexpression and knockout mouse models. This polarization is observed early in hematopoietic development, with an increase in common lymphoid progenitors (CLPs) and a decrease in all myeloid progenitor subsets in adult bone marrow. The pool size of multipotential progenitors (MPPs) is unchanged; however, in this report we observe by flow cytometry that polarized subsets of MPPs are changed in the absence of mirn23a. Additionally, in vitro culture of MPPs and sorted MPP transplants showed that these cells have decreased myeloid and increased lymphoid potential in vitro and in vivo. We investigated the mechanism by which mirn23a regulates hematopoietic differentiation and observed that mirn23a promotes myeloid development of hematopoietic progenitors through regulation of hematopoietic transcription factors and signaling pathways. Early transcription factors that direct the commitment of MPPs to CLPs (Ikzf1, Runx1, Satb1, Bach1 and Bach2) are increased in the absence of mirn23a miRNAs as well as factors that commit the CLP to the B cell lineage (FoxO1, Ebf1, and Pax5). Mirn23a appears to buffer transcription factor levels so that they do not stochastically reach a threshold level to direct differentiation. Intriguingly, mirn23a also inversely regulates the PI3 kinase (PI3K)/Akt and BMP/Smad signaling pathways. Pharmacological inhibitor studies, coupled with dominant active/dominant negative biochemical experiments, show that both signaling pathways are critical to mirn23a's regulation of hematopoietic differentiation. Lastly, consistent with mirn23a being a physiological inhibitor of B cell development, we observed that the essential B cell transcription factor EBF1 represses expression of mirn23a. In summary, our data demonstrates that mirn23a regulates a complex array of transcription and signaling pathways to modulate adult hematopoiesis.

Arid3b Is Critical for B Lymphocyte Development.

Arid3a and Arid3b belong to a subfamily of ARID (AT-rich interaction domain) transcription factors. The Arid family is involved in regulating chromatin accessibility, proliferation, and differentiation. Arid3a and Arid3b are closely related and share a unique REKLES domain that mediates their homo- and hetero-multimerization. Arid3a was originally isolated as a B cell transcription factor binding to the AT rich matrix attachment regions (MARS) of the immunoglobulin heavy chain intronic enhancer. Deletion of Arid3a results in a highly penetrant embryonic lethality with severe defects in erythropoiesis and hematopoietic stem cells (HSCs). The few surviving Arid3a-/- (<1%) animals have decreased HSCs and early progenitors in the bone marrow, but all mature lineages are normally represented in the bone marrow and periphery except for B cells. Arid3b-/- animals die around E7.5 precluding examination of hematopoietic development. So it is unclear whether the phenotype of Arid3a loss on hematopoiesis is dependent or independent of Arid3b. In this study we circumvented this limitation by also examining hematopoiesis in mice with a conditional allele of Arid3b. Bone marrow lacking Arid3b shows decreased common lymphoid progenitors (CLPs) and downstream B cell populations while the T cell and myeloid lineages are unchanged, reminiscent of the adult hematopoietic defect in Arid3a mice. Unlike Arid3a-/- mice, HSC populations are unperturbed in Arid3b-/- mice. This study demonstrates that HSC development is independent of Arid3b, whereas B cell development requires both Arid3a and Arid3b transcription factors.

The mirn23a microRNA cluster antagonizes B cell development.

Ablation of microRNA synthesis by deletion of the microRNA-processing enzyme Dicer has demonstrated that microRNAs are necessary for normal hematopoietic differentiation and function. However, it is still unclear which specific microRNAs are required for hematopoiesis and at what developmental stages they are necessary. This is especially true for immune cell development. We previously observed that overexpression of the products of the mirn23a gene (microRNA-23a, -24-2, and 27a) in hematopoietic progenitors increased myelopoiesis with a reciprocal decrease in B lymphopoiesis, both in vivo and in vitro. In this study, we generated a microRNA-23a, -24-2, and 27a germline knockout mouse to determine whether microRNA-23a, -24-2, and 27a expression was essential for immune cell development. Characterization of hematopoiesis in microRNA-23a, -24-2, and 27a-/- mice revealed a significant increase in B lymphocytes in both the bone marrow and the spleen, with a concomitant decrease in myeloid cells (monocytes/granulocytes). Analysis of the bone marrow progenitor populations revealed a significant increase in common lymphoid progenitors and a significant decrease in both bone marrow common myeloid progenitors and granulocyte monocyte progenitors. Gene-expression analysis of primary hematopoietic progenitors and multipotent erythroid myeloid lymphoid cells showed that microRNA-23a, -24-2, and 27a regulates essential B cell gene-expression networks. Overexpression of microRNA-24-2 target Tribbles homolog 3 can recapitulate the microRNA-23a, -24-2, and 27a-/- phenotype in vitro, suggesting that increased B cell development in microRNA-23a, -24-2, and 27a null mice can be partially explained by a Tribbles homolog 3-dependent mechanism. Data from microRNA-23a, -24-2, and 27a-/- mice support a critical role for this microRNA cluster in regulating immune cell populations through repression of B lymphopoiesis.

Evaluation of the mirn23a Cluster through an iTRAQ-based Quantitative Proteomic Approach.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that are implicated in a number of disease states. MiRNAs can exist as individual entities or may be clustered and transcribed as a single polycistron. The mirn23a cluster consists of three miRNAs: miR-23a, miR-24-2, and miR-27a. Although these miRNAs are transcribed together, they often exist at varying levels in the cell. Despite the fact that the mirn23a cluster is known to play a role in a number of diseases and developmental processes, few direct targets have been identified. In this study, we examined the effects of miR-23a, miR-24-2, miR-27a, or the mirn23a cluster overexpression on the proteome of 70Z/3 pre-B lymphoblast cells. Quantitative mass spectrometry using isobaric tags for relative and absolute quantification (iTRAQ) allowed for the global profiling of cell lines after miRNA overexpression. We identified a number of targets of each miRNA that contained predicted miRNA seed sequences and are likely direct targets. In addition, we discovered a cohort of shared miRNA targets and cluster targets, demonstrating the importance of studying miRNA clusters in their entirety.

E2A Antagonizes PU.1 Activity through Inhibition of DNA Binding.

Antagonistic interactions between transcription factors contribute to cell fate decisions made by multipotent hematopoietic progenitor cells. Concentration of the transcription factor PU.1 affects myeloid/lymphoid development with high levels of PU.1 directing myeloid cell fate acquisition at the expense of B cell differentiation. High levels of PU.1 may be required for myelopoiesis in order to overcome inhibition of its activity by transcription factors that promote B cell development. The B cell transcription factors, E2A and EBF, are necessary for commitment of multipotential progenitors and lymphoid primed multipotential progenitors to lymphocytes. In this report we hypothesized that factors required for early B cell commitment would bind to PU.1 and antagonize its ability to induce myeloid differentiation. We investigated whether E2A and/or EBF associate with PU.1. We observed that the E2A component, E47, but not EBF, directly binds to PU.1. Additionally E47 represses PU.1-dependent transactivation of the MCSFR promoter through antagonizing PU.1's ability to bind to DNA. Exogenous E47 expression in hematopoietic cells inhibits myeloid differentiation. Our data suggest that E2A antagonism of PU.1 activity contributes to its ability to commit multipotential hematopoietic progenitors to the lymphoid lineages.

MiR-24 is required for hematopoietic differentiation of mouse embryonic stem cells.

Overexpression of miRNA, miR-24, in mouse hematopoietic progenitors increases monocytic/ granulocytic differentiation and inhibits B cell development. To determine if endogenous miR-24 is required for hematopoiesis, we antagonized miR-24 in mouse embryonic stem cells (ESCs) and performed in vitro differentiations. Suppression of miR-24 resulted in an inability to produce blood and hematopoietic progenitors (HPCs) from ESCs. The phenotype is not a general defect in mesoderm production since we observe production of nascent mesoderm as well as mesoderm derived cardiac muscle and endothelial cells. Results from blast colony forming cell (BL-CFC) assays demonstrate that miR-24 is not required for generation of the hemangioblast, the mesoderm progenitor that gives rise to blood and endothelial cells. However, expression of the transcription factors Runx1 and Scl is greatly reduced, suggesting an impaired ability of the hemangioblast to differentiate. Lastly, we observed that known miR-24 target, Trib3, is upregulated in the miR-24 antagonized embryoid bodies (EBs). Overexpression of Trib3 alone in ESCs was able to decrease HPC production, though not as great as seen with miR-24 knockdown. These results demonstrate an essential role for miR-24 in the hematopoietic differentiation of ESCs. Although many miRNAs have been implicated in regulation of hematopoiesis, this is the first miRNA observed to be required for the specification of mammalian blood progenitors from early mesoderm.

Retroviral infection of murine embryonic stem cell derived embryoid body cells for analysis of hematopoietic differentiation.

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

ARID3B increases ovarian tumor burden and is associated with a cancer stem cell gene signature.

Ovarian cancer is the most deadly gynecological malignancy since most patients have metastatic disease at the time of diagnosis. Therefore, identification of critical pathways that contribute to ovarian cancer progression is necessary to yield novel therapeutic targets. Recently we reported that the DNA binding protein ARID3B is overexpressed in human ovarian tumors. To determine if ARID3B has oncogenic functions in vivo, ovarian cancer cell lines stably expressing ARID3B were injected intraperitoneally into nude mice. Overexpression of ARID3B increased tumor burden and decreased survival. To assess how ARID3B contributes to the increased tumor growth in vivo, we identified ARID3B induced genes in tumor ascites cells. ARID3B induced expression of genes associated with metastasis and cancer stem cells (CD44, LGR5, PROM1 (CD133), and Notch2). Moreover, ARID3B increased the number of CD133+ (a cancer stem cell marker) cells compared to control cells. The increase in CD133+ cells resulting from ARID3B expression was accompanied by enhanced paclitaxel resistance. Our data demonstrate that ARID3B boosts production CD133+ cells and increases ovarian cancer progression in vivo.

MiR-24 promotes the survival of hematopoietic cells.

The microRNA, miR-24, inhibits B cell development and promotes myeloid development of hematopoietic progenitors. Differential regulation of cell survival in myeloid and lymphoid cells by miR-24 may explain how miR-24's affects hematopoietic progenitors. MiR-24 is reported to regulate apoptosis, either positively or negatively depending on cell context. However, no role for miR-24 in regulating cell death has been previously described in blood cells. To examine miR-24's effect on survival, we expressed miR-24 via retrovirus in hematopoietic cells and induced cell death with cytokine or serum withdrawal. We observed that miR-24 enhanced survival of myeloid and B cell lines as well as primary hematopoietic cells. Additionally, antagonizing miR-24 with shRNA in hematopoietic cells made them more sensitive to apoptotic stimuli, suggesting miR-24 functions normally to promote blood cell survival. Since we did not observe preferential protection of myeloid over B cells, miR-24's pro-survival effect does not explain its promotion of myelopoiesis. Moreover, expression of pro-survival protein, Bcl-xL, did not mimic miR-24's impact on cellular differentiation, further supporting this conclusion. Our results indicate that miR-24 is a critical regulator of hematopoietic cell survival. This observation has implications for leukemogenesis. Several miRNAs that regulate apoptosis have been shown to function as either tumor suppressors or oncogenes during leukemogenesis. MiR-24 is expressed highly in primary acute myelogenous leukemia, suggesting that its pro-survival activity could contribute to the transformation of hematopoietic cells.