VSSP abrogates murine ovarian tumor-associated myeloid cell-driven immune suppression and induces M1 polarization in tumor-associated macrophages from ovarian cancer patients.

The ovarian tumor microenvironment (TME) is characterized by the accumulation of immunosuppressive tumor-associated macrophages (TAMs) and granulocytic cells. Very small size particles (VSSP), comprised of the ganglioside NAcGM3 and Neisseria meningitidis derived outer membrane vesicles, is being developed as a nanoparticulated modulator of innate immunity. Prior studies have shown that VSSP enhanced antigen-specific cytotoxic T cell responses and reduced the suppressive phenotype of splenic granulocytic cells in tumor-bearing mice. Here, we hypothesized that intraperitoneal VSSP would modify myeloid cell accumulation and phenotypes in the ovarian TME and abrogate suppressor function of TAMs and tumor-associated granulocytic cells. In the ID8 syngeneic model of epithelial ovarian cancer, VSSP reduced peritoneal TAMs and induced M1-like polarization in TAMs. In addition, VSSP stimulated peritoneal inflammation characterized by increased granulocytes and monocytes, including inflammatory monocytic cells. VSSP treatment resulted in peritoneal TAMs and granulocytic cells being less suppressive of ex vivo stimulated CD8+ T cell responses. VSSP alone and combined with anti-PD-1 modestly but significantly prolonged survival in tumor-bearing mice. In addition, ex vivo treatment with VSSP induced M1-like polarization in TAMs from patients with metastatic ovarian cancer and variably abrogated their suppressor phenotype. VSSP treatment also partially abrogated the induction of suppressor function in healthy donor neutrophils exposed to ascites supernatants from patients with ovarian cancer. Together, these results point to VSSP reprogramming myeloid responses resulting in abrogation of suppressive pathways and raise the potential for administration of VSSP into the TME to enhance anti-tumor immunity.

Dicer protein levels elevated by mild hyperthermia promote a pro-survival phenotype.

Cellular exposure to mild stress (39.5°C - 41.5°C) induces thermotolerance, rendering cells resistant to a subsequent heat shock (>42°C) insult. We found that mild hyperthermia at 39.5°C leads to elevations in dicer, a protein well-known for its role in microRNA processing and for its role in cellular stress responses. However, whether elevated dicer protein levels play a role in sustaining a thermotolerant phenotype has, to our knowledge, not been reported. Here we demonstrate that elevated dicer protein is linked to a thermotolerant phenotype in the cervical carcinoma cell line HeLa and in murine embryonic fibroblasts (MEF), and demonstrate that dicer plays a role in mediating PKR and eIF2α phosphorylation. These findings suggest that dicer's role in thermotolerance may be to relay signals to key ER stress pathway components. Moreover, utilizing a MEF cell line defective in microRNA processing, we suggest that dicer's influence on PKR and eIF2α phosphorylation is likely distinct from its microRNA processing role. ATF4 and CHOP are well characterized stress response factors proximal to eIF2α. Evidence is presented that elevated dicer protein in thermotolerant cells differentially modulates ATF4 and CHOP levels to promote a pro-survival phenotype. This work contributes new information on dicer's role in cellular stress responses by defining a pro-survival phenotype in heat stress resistant cells which is sustained, at least in part, by elevated dicer protein levels. Our results suggest an ancillary role for dicer in the cellular stress pathways activated by mild hyperthermia that is likely distinct from its role in microRNA processing.

The epigenetic regulation of Dicer and microRNA biogenesis by Panobinostat.

microRNAs (miRs) are small noncoding RNAs that regulate/fine tune many cellular protein networks by targeting mRNAs for either degradation or translational inhibition. Dicer, a type III endoribonuclease, is a critical component in miR biogenesis and is required for mature microRNA production. Abnormal Dicer expression occurs in numerous cancer types and correlates with poor patient prognosis. Recent reports have demonstrated that epigenetic agents, including histone deacetylase inhibitors (HDACi), may regulate Dicer and miR expression. HDACi are a class of epigenetic agents used to treat cancer, viral infections, and inflammatory disorders. However, little is known regarding the epigenetic regulation of miR biogenesis and function. We therefore investigated whether clinically successful HDACi modulated Dicer expression and found that Panobinostat, a clinically approved HDACi, enhanced Dicer expression via posttranscriptional mechanisms. Studies using proteasome inhibitors suggested that Panobinostat regulated the proteasomal degradation of Dicer. Further studies demonstrated that Panobinostat, despite increasing Dicer protein expression, decreased Dicer activity. This suggests that Dicer protein levels do not necessarily correlate with Dicer activity and mature miR levels. Taken together, we present evidence here that Panobinostat posttranscriptionally regulates Dicer/miR biogenesis and suggest Dicer as a potential therapeutic target in cancer.

The modulation of Dicer regulates tumor immunogenicity in melanoma.

MicroRNAs (miRs) are small non-coding RNAs that regulate most cellular protein networks by targeting mRNAs for translational inhibition or degradation. Dicer, a type III endoribonuclease, is a critical component in microRNA biogenesis and is required for mature microRNA production. Abnormal Dicer expression occurs in numerous cancer types and correlates with poor patient prognosis. For example, increased Dicer expression in melanoma is associated with more aggressive tumors (higher tumor mitotic index and depth of invasion) and poor patient prognosis. However, the role that Dicer plays in melanoma development and immune evasion remains unclear. Here, we report on a newly discovered relationship between Dicer expression and tumor immunogenicity. To investigate Dicer's role in regulating melanoma immunogenicity, Dicer knockdown studies were performed. We found that B16F0-Dicer deficient cells exhibited decreased tumor growth compared to control cells and were capable of inducing anti-tumor immunity. The decrease in tumor growth was abrogated in immunodeficient NSG mice and was shown to be dependent upon CD8+ T cells. Dicer knockdown also induced a more responsive immune gene profile in melanoma cells. Further studies demonstrated that CD8+ T cells preferentially killed Dicer knockdown tumor cells compared to control cells. Taken together, we present evidence which links Dicer expression to tumor immunogenicity in melanoma.

Dicer and microRNA expression in multiple sclerosis and response to interferon therapy.

Dysregulation of microRNA expression has been shown in multiple sclerosis (MS); however, the mechanisms underlying these changes, their response to therapy and the impact of microRNA changes in MS are not completely understood. Dicer mediates the cleavage of precursor microRNAs to mature microRNAs and is dysregulated in multiple pathologies. Having shown that interferons regulate Dicer in vitro, we hypothesized that MS patient IFNß1a treatment could potentially alter Dicer expression. Dicer mRNA and protein levels, as well as microRNA expression, were determined in MS patient and healthy control PBL. Acute responses to IFNß1a were assessed in 50 patients. We found that Dicer protein but not mRNA levels decreases in MS patients while both are selectively induced in patients responding well to IFNß1a. Potential microRNA biomarkers for relapsing remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS) and IFNß1a response are described. Contrasts in Dicer and microRNA expression levels between patient populations may offer insight into mechanisms underlying disease courses and responses to IFNß1a therapy. This work identifies Dicer regulation as both a potential mediator of MS pathology and a therapeutic target.

Dicer in immune cell development and function.

Dicer is an enzyme of the RNase III endoribonuclease family, which is crucial for RNA interference (RNAi) in eukaryotes. Dicer is a component of the protein machinery (the RNA Induced Silencing Complex [RISC]) which is involved in catalyzing the formation of mature microRNAs from their precursors in the process of microRNA biogenesis. RISC-associated microRNAs bind to specific sequences in the 3' untranslated region of cognate mRNAs largely through complementary base pairing, resulting in either translational inhibition and/or the degradation of a specific mRNA pool. MicroRNAs epigenetically regulate the cellular levels of receptors, transcription factors and signaling proteins that govern the developmental pathways and functions of multiple cellular processes. The pivotal role played by Dicer in microRNA formation has also piqued the interest of molecular immunologists who have sought to understand the biological relevance of microRNAs in the development and function of the immune system. Here, we review the major findings of these studies and provide an overview of the role of Dicer and microRNAs in immune cell development and function. Additionally, we highlight deficiencies in our knowledge and new research areas that may enhance our understanding of the role of Dicer and microRNAs in immunity.

Mild hyperthermia enhances the expression and induces oscillations in the Dicer protein.

PURPOSE: To investigate whether mild heat stress at 39.5°C altered Dicer protein and miRNA expression patterns in several cell types. METHODS: Multiple human and mouse cell types were cultured during the course of 9 h at temperatures from 37°C to 39.5°C. Dicer mRNA levels and microRNAs were quantified by TaqMan RT-qPCR assays and Dicer protein by western blotting. RESULTS: Dicer protein was substantially elevated on western analysis in response to heat stress at 39.5°C in the absence of significant changes in Dicer mRNA by RT-qPCR. CONCLUSIONS: Heat-induced regulation of Dicer expression occurs primarily post- transcriptionally, and the expression levels of Dicer protein are increased and often oscillate in response to fever-range hyperthermia in multiple mouse and human cells. Our studies suggest a potential role for Dicer and microRNAs in the response to mild thermal stress. Additional studies on the mechanisms involved in the stress-induced oscillations of Dicer protein and microRNAs will be of interest.

MHC class II regulation by epigenetic agents and microRNAs.

MicroRNAs have been shown to regulate gene expression both transcriptionally and translationally. Here, we examine evidence that various stresses regulate miRNAs which, in turn, regulate immune gene levels. Multiple studies are reviewed showing altered microRNA levels in normal cells under stress and in various disease states, including cancer. Unexpected was the finding that Dicer expression is altered by treatments with several agents, such as interferons and cortisone, employed in the treatment of immune disorders. Potential signal transduction pathways, including JAK/Stat, PI3K and PKR, that may regulate Dicer and microRNA levels in normal and stressed mammalian cells are discussed.

Restoration of immune response gene induction in trophoblast tumor cells associated with cellular senescence.

Trophoblast cells and many cancer cells that harbor foreign antigens may evade immunity by epigenetic silencing of key immune response genes, including MHC class I and II and CD40. Chromatin active agents, such as histone deacetylase inhibitors (HDACi), induce immune response gene expression but often the expression levels are low and the cells lack a robust antigen presentation response. We show here that pre-treatment of trophoblast cells and certain cancer cells with agents that activate stress pathways (Ras oncogene, PMA or H2O2) and induce senescence can substantially enhance the induction of immune response genes (MHC class II, CD40, MICA, MICB) by HDACi and restore a vigorous IFN-gamma response in trophoblast cells and tumor cells. These results could potentially impact the development of novel anti-cancer therapeutic strategies.

Dicer is regulated by cellular stresses and interferons.

The generation of microRNAs is dependent on the RNase III enzyme Dicer, the levels of which vary in different normal cells and in disease states. We demonstrate that Dicer protein expression in JAR trophoblast cells, and several other cell types, was inhibited by multiple stresses including reactive oxygen species, phorbol esters and the Ras oncogene. Additionally, double-stranded RNA and Type I interferons repress Dicer protein in contrast to IFN-gamma which induces Dicer. The effects of stresses and interferons are primarily post-transcriptional. The findings suggest that Dicer is a stress response component and identifies interferons as potentially important regulators of Dicer expression.

miRNA regulation of cytokine genes.

In this review we discuss specific examples of regulation of cytokine genes and focus on a new mechanism involving post-transcriptional regulation via miRNAs. The post-transcriptional regulation of cytokine genes via the destabilizing activity of AU-rich elements [AREs] and miRNAs is a pre-requisite for regulating the half-life of many cytokines and achieving the temporal and spatial distributions required for regulation of these genes.

Histone deacetylase regulation of immune gene expression in tumor cells.

Epigenetic modifications of chromatin, such as histone acetylation, are involved in repression of tumor antigens and multiple immune genes that are thought to facilitate tumor escape. The status of acetylation in a cell is determined by the balance of the activities of histone acetyltransferases and histone deacetylases. Inhibitors of histone deacetylase (HDACi) can enhance the expression of immunologically important molecules in tumor cells and HDACi treated tumor cells are able to induce immune responses in vitro and in vivo. Systemic HDACi are in clinical trails in cancer and also being used in several autoimmune disease models. To date, 18 HDACs have been reported in human cells and more than thirty HDACi identified, although only a few immune targets of these inhibitors have been identified. Here, we discuss the molecular pathways employed by HDACi and their potential role in inducing immune responses against tumors. We review data suggesting that selection of target specific HDACi and combinations with other agents and modalities, including those that activate stress pathways, may further enhance the efficacy of epigenetic therapies.

Histone deacetylase inhibitors induce TAP, LMP, Tapasin genes and MHC class I antigen presentation by melanoma cells.

Histone deacetylase inhibitors (HDACi), including trichostatin A (TSA) and valproic acid, can alter the acetylation of histones in chromatin and enhance gene transcription. Previously we demonstrated that HDACi-treated tumor cells are capable of presenting antigen via the MHC class II pathway. In this study, we show that treatment with HDACi enhances the expression of molecules (TAP1, TAP2, LMP2, LMP7, Tapasin and MHC class I) involved in antigen processing and presentation via the MHC class I pathway in melanoma cells. HDACi treatment of B16F10 cells also enhanced cell surface expression of class I and costimulatory molecules CD40 and CD86. Enhanced transcription of these genes is associated with a significant increase in direct presentation of whole protein antigen and MHC class I-restricted peptides by TSA-treated B16F10 cells. Our data indicate that epigenetic modification can convert a tumor cell to an antigen presenting cell capable of activating IFN-gamma secreting T cells via the class I pathway. These findings suggest that the abnormalities, observed in some tumors in the expression of MHC class I antigen processing and presentation molecules, may result from epigenetic repression.

MicroRNA targets in immune genes and the Dicer/Argonaute and ARE machinery components.

We studied 613 genes which regulate immunity and, utilizing predictive algorithms, identified 285 genes as microRNA (miRNA or miR) targets. Of these, approximately 250 are newly predicted gene-miR interactions. The frequency of predicted miRNA binding sites in immune gene 3'UTRs indicated preferential targeting of immune genes compared to the genome. Major targets include transcription factors, cofactors and chromatin modifiers whereas upstream factors, such as ligands and receptors (cytokines, chemokines and TLRs), were, in general, non-targets. About 10% of the immune genes were 'hubs' with eight or more different miRNAs predicted to target their 3'UTRs. Hubs were focused on certain key immune genes, such as BCL6, SMAD7, BLIMP1, NFAT5, EP300 and others. NF-kappaB and p53 do not themselves have binding sites for miRNAs but rather these pathways are targeted by miRNAs at downstream sites. MHC class II genes lacked miRNA targets but binding sites were identified in the CIITA gene and were shown experimentally to repress IFN-gamma-induced MHC class II activation. Unexpectedly, factors involved in regulating message stability via AU-rich elements (ARE) were heavily targeted. Moreover, multiple components involved in the generation and effector functions of miRNAs (Dicer and Argonautes) were themselves miRNA targets suggesting that a subset of miRNAs may indirectly control their own production as well as other miRNAs.

Spatial distribution of histone methylation during MHC class II expression.

We have previously reported that Major Histocompatibility Complex (MHC) class II can be induced by histone deacetylase inhibitors (HDACi) in the absence of class II transactivator (CIITA). Here we characterized the histone modifications associated with the CIITA-dependent (IFN-gamma induced) and -independent (HDACi induced) MHC class II expression. We demonstrate that both IFN-gamma and HDACi induced MHC class II expression exhibited enhanced histone H3, H4 acetylation and H3K4me3 at the MHC class II promoter while H3K9me3 was decreased. In contrast, high levels of H3K36me3 were detected at exons 3 and 5 but not at the promoter or the locus control region (LCR). Interestingly, high levels of H3K79me2 were only detected at the promoter and exon 3 of the B cell lines while the level remained low and unchanged despite active MHC class II expression induced by either IFN-gamma or HDACi treatment. Constitutive expression of the CIITA protein by stable transfection of a CIITA deficient B cell line restored the H3K79me2 to a level comparable to its cell of origin. This data demonstrates that, although regulated by different pathways, both IFN-gamma and HDACi treatments resulted in similar patterns of histone modifications and that HDACi induce both histone methylation and acetylation. In addition, the different spatial distribution of the lysine methylation markers along the gene suggests that these modifications play a distinctive role during different phases of the transcription process.

An epigenetic vaccine model active in the prevention and treatment of melanoma.

BACKGROUND: Numerous immune genes are epigenetically silenced in tumor cells and agents such as histone deacetylase inhibitors (HDACi), which reverse these effects, could potentially be used to develop therapeutic vaccines. The conversion of cancer cells to antigen presenting cells (APCs) by HDACi treatment could potentially provide an additional pathway, together with cross-presentation of tumor antigens by host APCs, to establish tumor immunity. METHODS: HDACi-treated B16 melanoma cells were used in a murine vaccine model, lymphocyte subset depletion, ELISpot and Cytotoxicity assays were employed to evaluate immunity. Antigen presentation assays, vaccination with isolated apoptotic preparations and tumorigenesis in MHC-deficient mice and radiation chimeras were performed to elucidate the mechanisms of vaccine-induced immunity. RESULTS: HDACi treatment enhanced the expression of MHC class II, CD40 and B7-1/2 on B16 cells and vaccination with HDACi-treated melanoma cells elicited tumor specific immunity in both prevention and treatment models. Cytotoxic and IFN-gamma-producing cells were identified in splenocytes and CD4+, CD8+ T cells and NK cells were all involved in the induction of immunity. Apoptotic cells derived from HDACi treatments, but not H2O2, significantly enhanced the effectiveness of the vaccine. HDACi-treated B16 cells become APCs in vitro and studies in chimeras defective in cross presentation demonstrate direct presentation in vivo and short-term but not memory responses and long-term immunity. CONCLUSION: The efficacy of this vaccine derives mainly from cross-presentation which is enhanced by HDACi-induced apoptosis. Additionally, epigenetic activation of immune genes may contribute to direct antigen presentation by tumor cells. Epigenetically altered cancer cells should be further explored as a vaccine strategy.

Epigenetic regulation of immune escape genes in cancer.

According to the concept of immune surveillance, the appearance of a tumor indicates that it has earlier evaded host defenses and subsequently must have escaped immunity to evolve into a full-blown cancer. Tumor escape mechanisms have focused mainly on mutations of immune and apoptotic pathway genes. However, data obtained over the past few years suggest that epigenetic silencing in cancer may be as frequent a cause of gene inactivation as are mutations. Here, we discuss the evidence that tumor immune evasion is mediated by non-mutational epigenetic events involving chromatin and that epigenetics collaborates with mutations in determining tumor progression. Since epigenetic changes are potentially reversible, the relative contribution of mutations and epigenetics, to the gene defects in any given tumor, may be a factor in determining the efficacy of treatments. We review new developments in basic chromatin mechanisms and in this context describe the rationale for the current use of epigenetic agents in cancer therapy and for a novel epigenetically generated tumor vaccine model. We emphasize that epigenetic cancer treatments are currently a 'blunt-sword' and suggest future directions for designing chromatin-based programs of potential value in the diagnosis and treatment of cancer.

The heat shock protein 90-CDC37 chaperone complex is required for signaling by types I and II interferons.

Interferon signaling pathways are critical to both innate and adaptive immunity. We have demonstrated here that the inhibition of heat shock protein 90 (Hsp90) functions by small interfering RNAs or chemical inhibitors blocking interferon-induced gene expression. Hsp90 was required for signal transducers and activators of transcription 1 phosphorylation, and in its absence, Janus kinase (JAK) 1/2 were degraded by the proteosome. JAK1 interacts with Hsp90 and the CDC37 co-chaperone, and both interactions are destabilized by Hsp90 inhibitors. The biological consequences were suggested by experiments showing that T cell activation by interferon-gamma-primed macrophages and the antiviral response of interferons required Hsp90. We conclude that JAK1/2 are client proteins of Hsp90 and that Hsp90 and CDC37 play a critical role in types I and II interferon pathways.

The major histocompatibility complex class II transactivator is differentially regulated by interferon-gamma and transforming growth factor-beta in microglial cells.

We evaluated the regulation of the major histocompatibility complex class II (MHC II) transactivator (CIITA) gene expression in two microglial cell lines, EOC2 and EOC20. We demonstrate that interferon-gamma (IFN-gamma) activates type III- and IV-CIITA mRNA and high levels of MHC II in EOC20. However, in EOC2 cells only low levels of type IV-CIITA mRNA and MHC II are detectable following IFN-gamma treatment. Transforming growth factor-beta1 (TGF-beta1) inhibits both type III- and IV-CIITA expression in EOC20 cells while, in EOC2 cells TGF-beta1 enhances IFN-gamma induced pIV-CIITA expression. Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, abrogates the TGF-beta1 mediated repression of the IFN-gamma induced CIITA in EOC20. Evidence is presented that the TG-interacting factor (TGIF), a co-repressor known to recruit HDACs, plays a role in determining the effects of TGF-beta1 on microglial cells.

Histone acetylation regulates the cell type specific CIITA promoters, MHC class II expression and antigen presentation in tumor cells.

The regulation of MHC class II expression by the class II transactivator (CIITA) is complex and differs in various cell types depending on the relative activity of three CIITA promoters. Here we show that, in plasma cell tumors, the deacetylase inhibitor trichostatin A (TSA) elicits PIII-CIITA but does not activate the IFN-gamma-inducible PIV-CIITA promoter. In trophoblast cells, all CIITA promoter types are constitutively silent and not induced by IFN-gamma or TSA treatment. TSA induction of PI-CIITA was restricted to macrophage and dendritic cell lines. In the Colon 26 tumor IFN-gamma induced endogenous PIV-CIITA but not PIII-CIITA while TSA activated class II in the apparent absence of CIITA. Reporter assays in Colon 26 showed that TSA induced PIII-CIITA but not PIV-CIITA. Transfection of a dominant negative CIITA plasmid in Colon 26 inhibited induction of class II by IFN-gamma but not TSA. Thus, the potential for both CIITA-dependent and -independent pathways of MHC induction exists within a single cell. Further evidence of CIITA-independent class II expression elicited by TSA was obtained using knockout mice with defects in CIITA, STAT-1alpha and IRF-1 expression. TSA treatment can also activate class II expression in mutant cell lines with deficiencies in signaling molecules, transcription factors and the BRG-1 cofactor that are required for IFN-gamma-induced CIITA expression. Importantly, after epigenetic activation by the deacetylase inhibitor, MHC class II is transported and displayed on the cell surface of a plasma cell tumor and it is converted to an efficient antigen presenting cell for protein and class II-peptide presentation.