Inactivation of the microRNA-183/96/182 cluster results in syndromic retinal degeneration

The microRNA-183/96/182 cluster is highly expressed in the retina and other sensory organs. To uncover its in vivo functions in the retina, we generated a knockout mouse model, designated “miR-183C^GT/GT,” using a gene-trap embryonic stem cell clone. We provide evidence that inactivation of the cluster results in early-onset and progressive synaptic defects of the photoreceptors, leading to abnormalities of scotopic and photopic electroretinograms with decreased b-wave amplitude as the primary defect and progressive retinal degeneration. In addition, inactivation of the miR-183/96/182 cluster resulted in global changes in retinal gene expression, with enrichment of genes important for synaptogenesis, synaptic transmission, photoreceptor morphogenesis, and phototransduction, suggesting that the miR-183/96/182 cluster plays important roles in postnatal functional differentiation and synaptic connectivity of photoreceptors.MicroRNAs (miRNAs) are small, endogenous, noncoding, regulatory RNAs and represent a newly recognized level of gene-expression regulation (1–4). miRNAs have unique expression profiles in the developing and adult retina and are involved in normal development and functions of the retina in all species studied so far (5–12). miRNAs are dysregulated in the retina of retinal degenerative mouse models, suggesting their potential involvement in retinal degeneration (13, 14). Conditional inactivation of dicer, an RNase III endonuclease required for miRNA maturation in cytosol (15), in the mouse retina resulted in alteration of retinal differentiation and optic-cup patterning, increased cell death, and disorganization of axons of retinal ganglion cells (16–19), suggesting that miRNAs are important for normal development and functions of the mammalian retina. However, in vivo functions of individual miRNAs in the retina still are largely unknown.Previously, we identified a highly conserved, intergenic, sensory organ-specific, paralogous miRNA cluster, the miR-183/96/182 cluster (hereafter, miR-183/96/182), contained within an ∼4-kb genomic segment on mouse chr6qA3.3 (8, 9). In the adult retina, miR-183/96/182 is expressed specifically in all photoreceptors and in the inner nuclear layer (8, 10). Developmentally, its expression is minimal in the embryonic retina but increases dramatically after birth and peaks in the adult retina, suggesting a role for miR-183/96/182 in maturation and normal functioning of the adult retina (8, 9). Additionally, expression of miR-183/96/182 has a diurnal pattern, suggesting a potential role in rhythmic functions of the retina (8, 9). Recently, miR-183/96/182 also was shown to be light responsive, independent of the circadian cycle (20). Targeted deletion of miR-182 alone in mouse did not result in a discernible phenotype, suggesting functional compensation by miR-183 and miR-96 (21). Point mutations of miR-96 were reported to result in progressive, nonsyndromic hearing loss in both human (22) and mouse (23); however, there was no apparent retinal phenotype, an observation that suggests miR-96 plays a major role in the inner ear but not in the retina (22–25). Finally, a recent report showed that knockdown of miR-183/96/182 in postmitotic rod photoreceptors in a miRNA-sponge transgenic mouse model resulted in increased susceptibility to light damage in the retina (26); however, no histological or functional defects of the retina were observed under normal lighting conditions (26). Thus, in vivo functions of miR-183/96/182 in the retina remain uncertain.To search for the in vivo functions of miR-183/96/182, we first dissected the genomic structure of the gene encoding miR-183/96/182 (hereafter referred to as “the miR-183/96/182 gene”) and characterized a gene-trap embryonic stem cell (ESC) clone (27–29) in which the gene-trap construct was inserted downstream of the first exon of the miR-183/96/182 gene, designated as “miR-183C^GT allele.” Using this ESC clone, we generated a mouse model, designated as “miR-183C^GT/GT,” in which the miR-183/96/182 gene is inactivated, and the β-geo cassette in the gene-trap construct reliably mirrors the endogenous expression patterns of miR-183/96/182.     

DNMT3a epigenetic program regulates the HIF-2α oxygen-sensing pathway and the cellular response to hypoxia

Epigenetic regulation of gene expression by DNA methylation plays a central role in the maintenance of cellular homeostasis. Here we present evidence implicating the DNA methylation program in the regulation of hypoxia-inducible factor (HIF) oxygen-sensing machinery and hypoxic cell metabolism. We show that DNA methyltransferase 3a (DNMT3a) methylates and silences the HIF-2α gene (EPAS1) in differentiated cells. Epigenetic silencing of EPAS1 prevents activation of the HIF-2α gene program associated with hypoxic cell growth, thereby limiting the proliferative capacity of adult cells under low oxygen tension. Naturally occurring defects in DNMT3a, observed in primary tumors and malignant cells, cause the unscheduled activation of EPAS1 in early dysplastic foci. This enables incipient cancer cells to exploit the HIF-2α pathway in the hypoxic tumor microenvironment necessary for the formation of cellular masses larger than the oxygen diffusion limit. Reintroduction of DNMT3a in DNMT3a-defective cells restores EPAS1 epigenetic silencing, prevents hypoxic cell growth, and suppresses tumorigenesis. These data support a tumor-suppressive role for DNMT3a as an epigenetic regulator of the HIF-2α oxygen-sensing pathway and the cellular response to hypoxia.Metazoan life is dependent upon the use of molecular oxygen for an array of metabolic processes. Tissue hypoxia occurs during periods of imbalance between oxygen supply and consumption. One of the primary cellular responses to hypoxia is the activation of the hypoxia-inducible factor (HIF) program (1–4). HIF consists of oxygen-regulated α-subunits HIF-1α and HIF-2α and a constitutively expressed β-subunit (HIF-β). In the presence of oxygen, a series of nonheme Fe(II)- and 2-oxoglutarate–dependent dioxygenase oxygen sensors, referred to as HIF prolylhydroxylases (HIF PHDs), promote the hydroxylation of key proline residues on the HIF-α subunits (5, 6). This serves as a recognition site for the von Hippel-Lindau (VHL) tumor-suppressor protein, which mediates ubiquitination and proteasomal degradation of HIF-1α and HIF-2α (7–9). Hypoxia inhibits HIF PHDs, allowing HIF-1α and HIF-2α to evade VHL recognition and assemble with HIF-β to produce the active heterodimeric HIF factor. Once activated, HIF-1α and HIF-2α cooperate through common and distinct pathways to regulate hypoxic gene expression and cellular adaptation to hypoxia (10).A notable feature of the HIF response is the differential expression pattern of HIF-1α and HIF-2α in normal tissues. HIF-1α mRNA is ubiquitous and constitutively expressed in adult cells. In stark contrast, HIF-2α mRNA is detected in a few cell types of adult tissues and is typically not expressed by epithelia (11). This suggests a physiological necessity to fine-tune the HIF program depending upon the cellular settings by negatively regulating the HIF-2α gene (EPAS1) upstream of the HIF oxygen-sensing enzymes. The negative regulation of EPAS1 is often compromised in cancers, as HIF-2α mRNA is observed in the vast majority of overt tumors (11–13). This is particularly evident in renal cancer. Elegant studies by the Maxwell group (13) and others (14) revealed that HIF-2α mRNA is absent in human kidney tubule epithelia but present in dysplastic foci of the nephron. In these incipient renal tumor cells, HIF-2α may function as an oncoprotein (15), collaborating with, or activating, multiple growth-promoting pathways including cancer stewards c-myc (16), ras (17), and EGFR (18, 19). Silencing of HIF-2α suppresses tumorigenesis of various genetically diverse cancers, further highlighting its central role in malignancy (16, 17, 20, 21), although this depends on the experimental context (22). Therefore, EPAS1 is silent in adult epithelia but undergoes unscheduled activation in several malignancies, driving proliferation in the hypoxic tumor microenvironment (23).A clue to the mechanisms involved in the unscheduled activation of EPAS1 during early tumorigenesis may reside in its promoter, which harbors an enrichment of cytosine and guanine bases that often serve as sites of DNA methylation and epigenetic gene silencing (24–27). Cytosine methylation is catalyzed by a family of DNA methyltransferases (DNMTs) including DNMT1, DNMT3a, and DNMT3b. DNMT1 maintains the methylation pattern from the template strand to the newly synthesized strand during DNA replication (28). DNMT3a and DNMT3b are de novo methyltransferases that establish postreplicative methylation patterns (29). Alterations in DNA methylation patterns are common in tumors and likely play a central role in aberrant gene expression that characterizes the malignant phenotype (26, 30, 31). This is particularly evident for DNMT3a, as recent studies have identified mutations in DNMT3a in patients with acute myeloid leukemia (32, 33) or down-regulation of DNMT3a mRNA in a variety of solid tumors (34). It is suggested that DNMT3a is a tumor-suppressor gene and that its mutation, or mRNA down-regulation, contributes to reducing global DNMT3a methyltransferase activity (35, 36). Currently, a key challenge is to link aberrant methylation profiles commonly observed in malignant lesions, including alterations in the DNMT3a epigenetic program, to genes that directly promote the tumorigenic phenotype.Here we show that DNMT3a methylates and silences EPAS1 in normal cells. Loss of DNMT3a observed in primary tumors and malignant cells causes unscheduled EPAS1 activation. This allows emerging cancer cells to exploit the HIF-2α program that facilitates cancer cell traverse of the hypoxic barrier and formation of tumors larger than the diffusion limit of oxygen. We suggest that the DNMT3a epigenetic program is a gatekeeper of the hypoxic cancer cell phenotype.     

Deletion of microRNA-155 reduces autoantibody responses and alleviates lupus-like disease in the Faslpr mouse

MicroRNA-155 (miR-155) regulates antibody responses and subsequent B-cell effector functions to exogenous antigens. However, the role of miR-155 in systemic autoimmunity is not known. Using the death receptor deficient (Fas^lpr) lupus-prone mouse, we show here that ablation of miR-155 reduced autoantibody responses accompanied by a decrease in serum IgG but not IgM anti-dsDNA antibodies and a reduction of kidney inflammation. MiR-155 deletion in Fas^lpr B cells restored the reduced SH2 domain-containing inositol 5′-phosphatase 1 to normal levels. In addition, coaggregation of the Fc γ receptor IIB with the B-cell receptor in miR-155^−/−-Fas^lpr B cells resulted in decreased ERK activation, proliferation, and production of switched antibodies compared with miR-155 sufficient Fas^lpr B cells. Thus, by controlling the levels of SH2 domain-containing inositol 5′-phosphatase 1, miR-155 in part maintains an activation threshold that allows B cells to respond to antigens.MicroRNA-155 (miR-155) plays a critical role in the generation of effective antibody responses to exogenous antigenic challenges in mice (1–3). MiR-155 levels have been reported to be elevated in B but low in T cells from patients with systemic lupus erythamosus (4), yet it is not known whether miR-155 controls autoimmune responses and the expression of related pathology.Mice harboring ubiquitous or B-cell-specific ablation of the death receptor Fas develop a severe lupus-like disease. B-cell-specific deletion of the death receptor (fas^−/−) fas^−/− mice develop an excessive germinal center (GC)-derived IgG autoantibody deposition in their kidneys and succumb to renal failure (5). It has been suggested that loss of tolerance in lpr mice results from the down-regulation of the low-affinity IgG inhibitory receptor FcγRIIB (Fc γ receptor IIB), thereby rendering their B cells incapable of terminating stimulatory signals delivered by autoantigen-containing immune complexes (6–8). However, the mechanisms whereby lack of FcγRIIB engagement would lead to autoimmunity, and whether additional factors contribute to autoimmunity, are still unclear.The SH2 domain-containing inositol 5′-phosphatase 1 (SHIP-1) phosphatase acts downstream of inhibitory cell-surface receptors (9–12), including the FcγRIIB, which is essential in opposing B-cell activation signals in mice and humans (13, 14). FcγRIIB inactivation has been implicated in the development of autoreactive GC B cells and plasma cells (15), as well as in the regulation of the persistence and longevity of bone marrow plasma cells (16). After coligation of the FcγRIIB with the B-cell receptor (BCR), FcγRIIB recruits SHIP-1 to the plasma membrane, where it negatively regulates cell survival, Ca²⁺-dependent effector functions, and ERK activation, thus controlling cell proliferation, anergy, and apoptosis (17–23). As a consequence of these wide-ranging activities, germ-line or B-cell-specific deletion of FcγRIIB or SHIP-1 in mice results in a severe lupus-like disease characterized by high-titer serum IgG antinuclear autoantibodies, lymphadenopathy, splenomegaly, renal failure, and increased mortality (23–27). MiR-155 has been reported to regulate SHIP-1 expression in mammalian myeloid and malignant B cells (28–31). However, it is not known whether SHIP-1 regulation by miR-155 affects GC reactions or peripheral tolerance during a protective immune response or in an autoimmune environment, such as that in Fas^lpr mice.To understand the role of miR-155 in autoimmunity, we crossed Fas^lpr mice with our bic/miR-155^−/− mice to generate miR-155^−/−-Fas^lpr animals. Here we demonstrate that deletion of miR-155 reduced serum IgG but not IgM anti-dsDNA autoantibody levels and kidney damage. Further, we show that the absence of miR-155 derepresses the expression of SHIP-1, thus mitigating B-cell activation, proliferation, and autoimmune responses. We provide evidence that miR-155 could be targeted to control autoimmunity and lupus nephritis.     

Potentiation of cytotoxic chemotherapy by growth hormone-releasing hormone agonists

The dismal prognosis of malignant brain tumors drives the development of new treatment modalities. In view of the multiple activities of growth hormone-releasing hormone (GHRH), we hypothesized that pretreatment with a GHRH agonist, JI-34, might increase the susceptibility of U-87 MG glioblastoma multiforme (GBM) cells to subsequent treatment with the cytotoxic drug, doxorubicin (DOX). This concept was corroborated by our findings, in vivo, showing that the combination of the GHRH agonist, JI-34, and DOX inhibited the growth of GBM tumors, transplanted into nude mice, more than DOX alone. In vitro, the pretreatment of GBM cells with JI-34 potentiated inhibitory effects of DOX on cell proliferation, diminished cell size and viability, and promoted apoptotic processes, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation assay, ApoLive-Glo multiplex assay, and cell volumetric assay. Proteomic studies further revealed that the pretreatment with GHRH agonist evoked differentiation decreasing the expression of the neuroectodermal stem cell antigen, nestin, and up-regulating the glial maturation marker, GFAP. The GHRH agonist also reduced the release of humoral regulators of glial growth, such as FGF basic and TGFβ. Proteomic and gene-expression (RT-PCR) studies confirmed the strong proapoptotic activity (increase in p53, decrease in v-myc and Bcl-2) and anti-invasive potential (decrease in integrin α3) of the combination of GHRH agonist and DOX. These findings indicate that the GHRH agonists can potentiate the anticancer activity of the traditional chemotherapeutic drug, DOX, by multiple mechanisms including the induction of differentiation of cancer cells.Glioblastoma multiforme (GBM) is one of the most aggressive human cancers, and the afflicted patients inevitably succumb. The dismal outcome of this malignancy demands great efforts to find improved methods of treatment (1). Many compounds have been synthesized in our laboratory in the past few years that have proven to be effective against diverse malignant tumors (2–14). These are peptide analogs of hypothalamic hormones: luteinizing hormone-releasing hormone (LHRH), growth hormone-releasing hormone (GHRH), somatostatin, and analogs of other neuropeptides such as bombesin and gastrin-releasing peptide. The receptors for these peptides have been found to be widely distributed in the human body, including in many types of cancers (2–14). The regulatory functions of these hypothalamic hormones and other neuropeptides are not confined to the hypothalamo–hypophyseal system or, even more broadly, to the central nervous system (CNS). In particular, GHRH can induce the differentiation of ovarian granulosa cells and other cells in the reproductive system and function as a growth factor in various normal tissues, benign tumors, and malignancies (2–4, 6, 11, 14–18). Previously, we also reported that antagonistic cytototoxic derivatives of some of these neuropeptides are able to inhibit the growth of several malignant cell lines (2–14).Our earlier studies showed that treatment with antagonists of LHRH or GHRH rarely effects complete regression of glioblastoma-derived tumors (5, 7, 10, 11). Previous studies also suggested that growth factors such as EGF or agonistic analogs of LHRH serving as carriers for cytotoxic analogs and functioning as growth factors may sensitize cancer cells to cytotoxic treatments (10, 19) through the activation of maturation processes. We therefore hypothesized that pretreatment with one of our GHRH agonists, such as JI-34 (20), which has shown effects on growth and differentiation in other cell lines (17, 18, 21, 22), might decrease the pluripotency and the adaptability of GBM cells and thereby increase their susceptibility to cytotoxic treatment.In vivo, tumor cells were implanted into athymic nude mice, tumor growth was recorded weekly, and final tumor mass was measured upon autopsy. In vitro, proliferation assays were used for the determination of neoplastic proliferation and cell growth. Changes in stem (nestin) and maturation (GFAP) antigen expression was evaluated with Western blot studies in vivo and with immunocytochemistry in vitro. The production of glial growth factors (FGF basic, TGFβ) was verified by ELISA. Further, using the Human Cancer Pathway Finder real-time quantitative PCR, numerous genes that play a role in the development of cancer were evaluated. We placed particular emphasis on the measurement of apoptosis, using the ApoLive-Glo Multiplex Assay kit and by detection of the expression of the proapoptotic p53 protein. This overall approach permitted the evaluation of the effect of GHRH agonist, JI-34, on the response to chemotherapy with doxorubicin.     

Insulin growth factor signaling is regulated by microRNA-486, an underexpressed microRNA in lung cancer

MicroRNAs (miRNAs) are small 19- to 24-nt noncoding RNAs that have the capacity to regulate fundamental biological processes essential for cancer initiation and progression. In cancer, miRNAs may function as oncogenes or tumor suppressors. Here, we conducted global profiling for miRNAs in a cohort of stage 1 nonsmall cell lung cancers (n = 81) and determined that miR-486 was the most down-regulated miRNA in tumors compared with adjacent uninvolved lung tissues, suggesting that miR-486 loss may be important in lung cancer development. We report that miR-486 directly targets components of insulin growth factor (IGF) signaling including insulin-like growth factor 1 (IGF1), IGF1 receptor (IGF1R), and phosphoinositide-3-kinase, regulatory subunit 1 (alpha) (PIK3R1, or p85a) and functions as a potent tumor suppressor of lung cancer both in vitro and in vivo. Our findings support the role for miR-486 loss in lung cancer and suggest a potential biological link to p53.Lung cancer is the number one cause of cancer related deaths with a frustratingly poor 5-y survival rate. Despite these statistics, the advent of both early detection and targeted therapies provide support for improved outcomes in the not-too-distant future. Therapies targeted toward specific mutations including Epidermal Growth Factor Receptor (EGFR) and Anaplastic Lymphoid Kinase (ALK) have proven to be of clinical benefit in selected subgroups of patients (1, 2). However, these mutations represent only two of a multitude of mutations in nonsmall cell lung cancer (NSCLC) that could eventually be leveraged for therapeutic purposes. MicroRNAs (miRNAs), a family of short endogenous noncoding RNAs, harbor critical functions in the initiation and progression of a variety of solid and hematological malignancies (3–5). Our laboratory made the early observation that miR-15/16 expression is down-regulated in the majority of cases of chronic lymphocytic leukemia (6). Recently, miRNAs have emerged in NSCLC as both diagnostic and prognostic biomarkers (7, 8). However, the basic mechanisms by which miRNAs function as tumor suppressors or oncogenes in NSCLC and their regulatory factors are known for only a handful of miRNAs.The Insulin Growth Factor (IGF) pathway is activated in several malignancies including NSCLC (9). In addition, signaling through IGF1R is essential for normal development and growth. Through downstream activation of both PI3K/AKT/mTOR and RAS/RAF/MAP kinase, IGF1R controls cell survival and proliferation respectively (10, 11). Several ongoing clinical trials have focused on strategies for directed targeting of IGF signaling but have had mixed results (12, 13). Despite these results, and given the fundamental role of this axis in tumor initiation and progression, IGF signaling continues to be the focus of investigation particularly for the development of targeted therapeutics in selected subgroups of patients. A few studies to date have validated components of IGF signaling as targets for miRNAs. For example, in breast cancer, miR-148a and miR-152 target both IGF1R and Insulin Receptor Substrate (IRS-1) leading to a reduction in both tumor proliferation and angiogenesis (14). MiR-145 also functions as a potent tumor suppressor in both colon and hepatocellular carcinoma. Components of IGF signaling including IRS-1, IRS-2, and IGF1R have been validated as targets of miR-122 and miR-145 (15–17). Located within the ankyrin-1 gene (18), miR-486 is deregulated across several solid malignancies (19) including osteosarcoma (20), pancreatic cancer (21), gastric cancer (22), and lung cancer (23). Interestingly, two recent investigations identified miR-486 as a potential noninvasive biomarker for the detection of lung cancer (24–26). To date, investigators have identified a few potential targets for miR-486, including the antiapoptotic OLFM4 (22), SIRT1 (27), and the tumor suppressor PTEN (18). However, the mechanistic role for miR-486 as either an oncogene or tumor suppressor particularly in lung cancer remains largely unknown. In the current study, we conducted a high-throughput miRNA array in a cohort of stage 1 NSCLC (n = 81) and determined that miR-486 was the most decreased miRNA compared with adjacent uninvolved lung tissues. We subsequently demonstrated that miR-486 functions as a potent suppressor of cellular proliferation, migratory capacity, and tumor growth both in vitro and in vivo. Furthermore, we validated components of IGF signaling including IGF1, IGF1R, and p85α as targets of miR-486. Lastly, we determined that the biological effects of miR-486 are partially dependent upon intact p53.     

Highly penetrative,drug-loaded nanocarriers improve treatment of glioblastoma

Current therapy for glioblastoma multiforme is insufficient, with nearly universal recurrence. Available drug therapies are unsuccessful because they fail to penetrate through the region of the brain containing tumor cells and they fail to kill the cells most responsible for tumor development and therapy resistance, brain cancer stem cells (BCSCs). To address these challenges, we combined two major advances in technology: (i) brain-penetrating polymeric nanoparticles that can be loaded with drugs and are optimized for intracranial convection-enhanced delivery and (ii) repurposed compounds, previously used in Food and Drug Administration-approved products, which were identified through library screening to target BCSCs. Using fluorescence imaging and positron emission tomography, we demonstrate that brain-penetrating nanoparticles can be delivered to large intracranial volumes in both rats and pigs. We identified several agents (from Food and Drug Administration-approved products) that potently inhibit proliferation and self-renewal of BCSCs. When loaded into brain-penetrating nanoparticles and administered by convection-enhanced delivery, one of these agents, dithiazanine iodide, significantly increased survival in rats bearing BCSC-derived xenografts. This unique approach to controlled delivery in the brain should have a significant impact on treatment of glioblastoma multiforme and suggests previously undescribed routes for drug and gene delivery to treat other diseases of the central nervous system.Of the ∼40,000 people diagnosed with primary brain tumors in the United States each year, an estimated 15,000 have glioblastoma multiforme (GBM), a World Health Organization grade IV malignant glioma (1). Despite considerable research efforts, the prognosis for GBM remains poor: median survival with standard-of-care therapy (surgery, systemic chemotherapy with temozolomide, and radiation) is 14.6 mo (2) and 5-y survival is 9.8% (3), with the vast majority of GBMs recurring within 2 cm of the original tumor focus (4). Histopathologically, GBM is characterized by its infiltrative nature and cellular heterogeneity, leading to a number of challenges that must be overcome by any presumptive therapy.The blood–brain barrier (BBB) is a major obstacle to treating GBM (5). It is estimated that over 98% of small-molecule drugs and ∼100% of large-molecule drugs or genes do not cross the BBB (6). Delivery of chemotherapeutics to the brain can be potentially achieved by using nanocarriers engineered for receptor-mediated transport across the BBB (7, 8), but the percentage of i.v. administered particles that enter the brain is low. It is not yet clear whether sufficient quantities of drug can be delivered by systemically administered nanoparticles to make this a useful method for treating tumors in the human brain. An alternate approach is to bypass the BBB: Clinical trials have demonstrated that the BBB can be bypassed with direct, locoregional delivery of therapeutic agents. For example, local implantation of a drug-loaded biodegradable polymer wafer (presently marketed as Gliadel), which slowly releases carmustine over a prolonged period, is a safe method for treating GBM. However, use of the Gliadel wafer results in only modest improvements in patient survival, typically 2 mo (9, 10). In prior work we showed that these wafers produce high interstitial drug concentrations in the tissue near the implant, but—because drugs move from the implant into the tissue by diffusion—penetration into tissue is limited to ∼1 mm, which could limit their efficacy (11, 12).We hypothesize that treatment of GBM can be improved by attention to three challenges: (i) enhancing the depth of penetration of locally delivered therapeutic agents, (ii) providing for long-term release of active agents, and (iii) delivering agents that are known to be effective against the cells that are most important in tumor recurrence. The first challenge can be addressed by convection-enhanced delivery (CED), in which agents are infused into the brain under a positive pressure gradient, creating bulk fluid movement in the brain interstitium (13). Recent clinical trials show that CED is safe and feasible (14–16), but CED alone is not sufficient to improve GBM treatment. For example, CED of a targeted toxin in aqueous suspension failed to show survival advantages over Gliadel wafers (14, 17). Although CED of drugs in solution results in increased penetration, most drugs have short half-lives in the brain and, as a result, they disappear soon after the infusion stops (17, 18). Loading of agents into nanocarriers—such as liposomes, micelles, dendrimers, or nanoparticles—can protect them from clearance. Significant progress has been made in CED of liposomes to the brain (19), although it is not clear that liposomes offer the advantage of long-term release. By contrast, CED of polymeric nanoparticles, such as nanoparticles made of poly(lactide-coglycolide) (PLGA), offers the possibility of controlled agent release. However, CED of PLGA nanoparticles, which are typically 100–200 nm in diameter, has been limited by the failure of particles to move by convection through the brain interstitial spaces (20–23), which are 38–64 nm in normal brain (24) and 7–100 nm in regions with tumor (25). Therefore, to overcome the first and second challenges, it is necessary to synthesize polymer nanocarriers that are much smaller than conventional particles and still capable of efficient drug loading and controlled release. We report here reliable methods for making PLGA nanoparticles with these characteristics.Drug developers have long been frustrated by the BBB, which severely limits the types of agents that can be tested for activity in the brain. We reasoned that creation of safe, versatile, brain-penetrating nanocarriers should enable direct testing of novel agents that address the complexity of GBM biology. For example, cells isolated from distinct regions of a given GBM bear grossly different expression signatures but seem to arise from a common progenitor (26): A small subpopulation of these progenitors drives tumor progression, promotes angiogenesis, and influences tumor cell migration (27–30). These cells have features of primitive neural stem cells and are called brain cancer stem cells (BCSCs) (29, 31–37). BCSCs, many of which are marked by CD133 (PROM1), are resistant to conventional drugs (28, 38), including carboplatin, cisplatin, paclitaxel, doxorubicin, vincristine, methotrexate, and temozolomide (39–42), as well as radiotherapy (29). These observations suggest that agents that affect BCSCs are more likely to lead to a cure for GBM (28, 38, 43, 44). Therefore, to illustrate the translational potential of brain-penetrating nanoparticles, we conducted a screen of ∼2,000 compounds that were previously used in Food and Drug Administration (FDA)-approved products for their ability to inhibit patient-derived BCSCs, encapsulated the best agents to emerge from the screen into brain-penetrating PLGA nanoparticles, and administered these nanocarriers by CED in a BCSC-derived xenograft model of GBM.     

Neofunction of ACVR1 in fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification. FOP patients harbor point mutations in ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP). Two mechanisms of mutated ACVR1 (FOP-ACVR1) have been proposed: ligand-independent constitutive activity and ligand-dependent hyperactivity in BMP signaling. Here, by using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs), we report a third mechanism, where FOP-ACVR1 abnormally transduces BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling but not BMP signaling. Activin-A enhanced the chondrogenesis of induced mesenchymal stromal cells derived from FOP-iPSCs (FOP-iMSCs) via aberrant activation of BMP signaling in addition to the normal activation of TGF-β signaling in vitro, and induced endochondral ossification of FOP-iMSCs in vivo. These results uncover a novel mechanism of extraskeletal bone formation in FOP and provide a potential new therapeutic strategy for FOP.Heterotopic ossification (HO) is defined as bone formation in soft tissue where bone normally does not exist. It can be the result of surgical operations, trauma, or genetic conditions, one of which is fibrodysplasia ossificans progressiva (FOP). FOP is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification (1–6). The responsive mutation for classic FOP is 617G > A (R206H) in the intracellular glycine- and serine-rich (GS) domain (7) of ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP) (8–10). ACVR1 mutations in atypical FOP patients have been found also in other amino acids of the GS domain or protein kinase domain (11, 12). Regardless of the mutation site, mutated ACVR1 (FOP-ACVR1) has been shown to activate BMP signaling without exogenous BMP ligands (constitutive activity) and transmit much stronger BMP signaling after ligand stimulation (hyperactivity) (12–25).To reveal the molecular nature of how FOP-ACVR1 activates BMP signaling, cells overexpressing FOP-ACVR1 (12–20), mouse embryonic fibroblasts derived from Alk2^R206H/+ mice (21, 22), and cells from FOP patients, such as stem cells from human exfoliated deciduous teeth (23), FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) (24, 25) and induced mesenchymal stromal cells (iMSCs) from FOP-iPSCs (FOP-iMSCs) (26) have been used as models. Among these cells, Alk2^R206H/+ mouse embryonic fibroblasts and FOP-iMSCs are preferred because of their accessibility and expression level of FOP-ACVR1 using an endogenous promoter. In these cells, however, the constitutive activity and hyperactivity is not strong (within twofold normal levels) (22, 26). In addition, despite the essential role of BMP signaling in development (27–31), the pre- and postnatal development and growth of FOP patients are almost normal, and HO is induced in FOP patients after physical trauma and inflammatory response postnatally, not at birth (1–6). These observations led us to hypothesize that FOP-ACVR1 abnormally responds to noncanonical BMP ligands induced by trauma or inflammation.Here we show that FOP-ACVR1 transduced BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling (10, 32–34) and contributes to inflammatory responses (35, 36). Our in vitro and in vivo data indicate that activation of TGF-β and aberrant BMP signaling by Activin-A in FOP-cells is one cause of HO in FOP. These results suggest a possible application of anti–Activin-A reagents as a new therapeutic tool for FOP.