Agnieszka Bronisz

MicroRNA-451 Regulates LKB1/AMPK Signaling and Allows Adaptation to Metabolic Stress in Glioma Cells

To sustain tumor growth, cancer cells must be able to adapt to fluctuations in energy availability. We have identified a single microRNA that controls glioma cell proliferation, migration, and responsiveness to glucose deprivation. Abundant glucose allows relatively high miR-451 expression, promoting cell growth. In low glucose, miR-451 levels decrease, slowing proliferation but enhancing migration and survival. This allows cells to survive metabolic stress and seek out favorable growth conditions. In glioblastoma patients, elevated miR-451 is associated with shorter survival. The effects of miR-451 are mediated by LKB1, which it represses through targeting its binding partner, CAB39 (MO25 alpha). Overexpression of miR-451 sensitized cells to glucose deprivation, suggesting that its downregulation is necessary for robust activation of LKB1 in response to metabolic stress. Thus, miR-451 is a regulator of the LKB1/AMPK pathway, and this may represent a fundamental mechanism that contributes to cellular adaptation in response to altered energy availability.

Fusion Tyrosine Kinases Induce Drug Resistance by Stimulation of Homology-Dependent Recombination Repair, Prolongation of G 2 /M Phase, and Protection from Apoptosis

ABSTRACT Fusion tyrosine kinases (FTKs) such as BCR/ABL, TEL/ABL, TEL/JAK2, TEL/PDGFβR, TEL/TRKC(L), and NPM/ALK arise from reciprocal chromosomal translocations and cause acute and chronic leukemias and non-Hodgkins lymphoma. FTK-transformed cells displayed drug resistance against the cytostatic drugs cisplatin and mitomycin C. These cells were not protected from drug-mediated DNA damage, implicating activation of the mechanisms preventing DNA damage-induced apoptosis. Various FTKs, except TEL/TRKC(L), can activate STAT5, which may be required to induce drug resistance. We show that STAT5 is essential for FTK-dependent upregulation of RAD51, which plays a central role in homology-dependent recombinational repair (HRR) of DNA double-strand breaks (DSBs). Elevated levels of Rad51 contributed to the induction of drug resistance and facilitation of the HRR in FTK-transformed cells. In addition, expression of antiapoptotic protein Bcl-xL was enhanced in cells transformed by the FTKs able to activate STAT5. Moreover, cells transformed by all examined FTKs displayed G2/M delay upon drug treatment. Individually, elevated levels of Rad51, Bcl-xL, or G2/M delay were responsible for induction of a modest drug resistance. Interestingly, combination of these three factors in nontransformed cells induced drug resistance of a magnitude similar to that observed in cells expressing FTKs activating STAT5. Thus, we postulate that RAD51-dependent facilitation of DSB repair, antiapoptotic activity of Bcl-xL, and delay in progression through the G2/M phase work in concert to induce drug resistance in FTK-positive leukemias and lymphomas.

MITF and PU.1 Recruit p38 MAPK and NFATc1 to Target Genes during Osteoclast Differentiation

Transcription factors NFATc1, PU.1, and MITF collaborate to regulate specific genes in response to colony-stimulating factor-1 (CSF-1) and receptor activator of NF-κB ligand (RANKL) signaling during osteoclast differentiation. However, molecular details concerning timing and mechanism of specific events remain ill-defined. In bone marrow-derived precursors, CSF-1 alone promoted assembly of MITF-PU.1 complexes at osteoclast target gene promoters like cathepsin K and acid 5 phosphatase without increasing gene expression. The combination of RANKL and CSF-1 concurrently increased the levels of MAPK-phosphorylated forms of MITF, p38 MAPK, and SWI/SNF chromatin-remodeling complexes bound to these target promoters and markedly increased expression of the genes. NFATc1 was subsequently recruited to complexes at the promoters during terminal stages of osteoclast differentiation. Genetic analysis of Mitf and Pu.1 in mouse models supported the critical interaction of these genes in osteoclast differentiation. The results define MITF and PU.1 as nuclear effectors that integrate CSF-1/RANKL signals during osteoclast differentiation to initiate expression of target genes, whereas a complex that includes NFATc1 may act to maintain target gene expression in differentiated cells.

microRNA-451: A conditional switch controlling glioma cell proliferation and migration

Glioblastoma, the most common and aggressive primary brain tumor, is rapidly growing, and highly infiltrative. Incomplete knowledge of the molecular biology, genetics, causes and cellular origin of these tumors may limit the development of improved therapeutics. A major and fundamental advance in recent years has been the identification of microRNAs as highly conserved regulators of gene expression. Here we will discuss further our recently published data on the role of miR-451 in the biology of glioblastoma. We initially identified miR-451 due to its downregulation in a glioma cell migration assay. We then found that by targeting the LKB1 kinase complex miR-451 suppresses the activity of downstream protein kinases including the major energy biosensor AMPK. MiR-451 levels are regulated by glucose; under conditions of abundant energy miR-451 expression is high, and the suppression of AMPK signaling allows cells to maintain elevated proliferation rates via unrestrained mTOR activation. Under conditions of glucose withdrawal, miR-451 downregulation is necessary for AMPK pathway activation, leading to suppressed proliferation rates, increased cell survival, and migration. We also identified a potential feedback loop between LKB1 and miR-451, which allows a sustained and robust response to glucose deprivation. This data will be discussed in the context of potential biological significance and therapeutic implications.

Extracellular Vesicles Modulate the Glioblastoma Microenvironment via a Tumor Suppression Signaling Network Directed by miR-1

Extracellular vesicles have emerged as important mediators of intercellular communication in cancer, including by conveying tumor-promoting microRNAs between cells, but their regulation is poorly understood. In this study, we report the findings of a comparative microRNA profiling and functional analysis in human glioblastoma that identifies miR-1 as an orchestrator of extracellular vesicle function and glioblastoma growth and invasion. Ectopic expression of miR-1 in glioblastoma cells blocked in vivo growth, neovascularization, and invasiveness. These effects were associated with a role for miR-1 in intercellular communication in the microenvironment mediated by extracellular vesicles released by cancer stem-like glioblastoma cells. An extracellular vesicle-dependent phenotype defined by glioblastoma invasion, neurosphere growth, and endothelial tube formation was mitigated by loading miR-1 into glioblastoma-derived extracellular vesicles. Protein cargo in extracellular vesicles was characterized to learn how miR-1 directed extracellular vesicle function. The mRNA encoding Annexin A2 (ANXA2), one of the most abundant proteins in glioblastoma-derived extracellular vesicles, was found to be a direct target of miR-1 control. In addition, extracellular vesicle-derived miR-1 along with other ANXA2 extracellular vesicle networking partners targeted multiple pro-oncogenic signals in cells within the glioblastoma microenvironment. Together, our results showed how extracellular vesicle signaling promotes the malignant character of glioblastoma and how ectopic expression of miR-1 can mitigate this character, with possible implications for how to develop a unique miRNA-based therapy for glioblastoma management.

Eos, MITF, and PU.1 Recruit Corepressors to Osteoclast-Specific Genes in Committed Myeloid Progenitors

ABSTRACT Transcription factors MITF and PU.1 collaborate to increase expression of target genes like cathepsin K (Ctsk) and acid phosphatase 5 (Acp5) during osteoclast differentiation. We show that these factors can also repress transcription of target genes in committed myeloid precursors capable of forming either macrophages or osteoclasts. The direct interaction of MITF and PU.1 with the zinc finger protein Eos, an Ikaros family member, was necessary for repression of Ctsk and Acp5. Eos formed a complex with MITF and PU.1 at target gene promoters and suppressed transcription through recruitment of corepressors CtBP (C-terminal binding protein) and Sin3A, but during osteoclast differentiation, Eos association with Ctsk and Acp5 promoters was significantly decreased. Subsequently, MITF and PU.1 recruited coactivators to these target genes, resulting in robust expression of target genes. Overexpression of Eos in bone marrow-derived precursors disrupted osteoclast differentiation and selectively repressed transcription of MITF/PU.1 targets, while small interfering RNA knockdown of Eos resulted in increased basal expression of Ctsk and Acp5. This work provides a mechanism to account for the modulation of MITF and PU.1 activity in committed myeloid progenitors prior to the initiation of osteoclast differentiation in response to the appropriate extracellular signals.

MicroRNA-128 coordinately targets Polycomb Repressor Complexes in glioma stem cells

BACKGROUND The Polycomb Repressor Complex (PRC) is an epigenetic regulator of transcription whose action is mediated by 2 protein complexes, PRC1 and PRC2. PRC is oncogenic in glioblastoma, where it is involved in cancer stem cell maintenance and radioresistance. METHODS We used a set of glioblastoma patient samples, glioma stem cells, and neural stem cells from a mouse model of glioblastoma. We characterized gene/protein expression and cellular phenotypes by quantitative PCR/Western blotting and clonogenic, cell-cycle, and DNA damage assays. We performed overexpression/knockdown studies by lentiviral infection and microRNA/small interfering RNA oligonucleotide transfection. RESULTS We show that microRNA-128 (miR-128) directly targets mRNA of SUZ12, a key component of PRC2, in addition to BMI1, a component of PRC1 that we previously showed as a target as well. This blocks the partially redundant functions of PRC1/PRC2, thereby significantly reducing PRC activity and its associated histone modifications. MiR-128 and SUZ12/BMI1 show opposite expression in human glioblastomas versus normal brain and in glioma stemlike versus neural stem cells. Furthermore, miR-128 renders glioma stemlike cells less radioresistant by preventing the radiation-induced expression of both PRC components. Finally, miR-128 expression is significantly reduced in neural stem cells from the brain of young, presymptomatic mice in our mouse model of glioblastoma. This suggests that loss of miR-128 expression in brain is an early event in gliomagenesis. Moreover, knockdown of miR-128 expression in nonmalignant mouse and human neural stem cells led to elevated expression of PRC components and increased clonogenicity. CONCLUSIONS MiR-128 is an important suppressor of PRC activity, and its absence is an early event in gliomagenesis.

SRGAP1 Is a Candidate Gene for Papillary Thyroid Carcinoma Susceptibility

BACKGROUND Papillary thyroid carcinoma (PTC) shows high heritability, yet efforts to find predisposing genes have been largely negative. OBJECTIVES The objective of this study was to identify susceptibility genes for PTC. METHODS A genome-wide linkage analysis was performed in 38 families. Targeted association study and screening were performed in 2 large cohorts of PTC patients and controls. Candidate DNA variants were tested in functional studies. RESULTS Linkage analysis and association studies identified the Slit-Robo Rho GTPase activating protein 1 gene (SRGAP1) in the linkage peak as a candidate gene. Two missense variants, Q149H and A275T, localized in the Fes/CIP4 homology domain segregated with the disease in 1 family each. One missense variant, R617C, located in the RhoGAP domain occurred in 1 family. Biochemical assays demonstrated that the ability to inactivate CDC42, a key function of SRGAP1, was severely impaired by the Q149H and R617C variants. CONCLUSIONS Our findings suggest that SRGAP1 is a candidate gene in PTC susceptibility. SRGAP1 is likely a low-penetrant gene, possibly of a modifier type.

Belonging to a network—microRNAs, extracellular vesicles, and the glioblastoma microenvironment

The complexity of glioblastoma multiforme (GBM) and its distinct pathophysiology belong to a unique brain microenvironment and its cellular interactions. Despite extensive evidence of a role for microRNAs in GBM cells, little is known about microRNA-dependent communication between different cellular compartments of the microenvironment that may contribute to the tumor phenotype. While the majority of microRNAs are found intracellularly, a significant number of microRNAs have been observed outside of cells, often encapsulated in secreted extracellular vesicles (EVs). The function of these circulating/secreted microRNAs has not been explored in the context of the brain tumor microenvironment. Establishing how microRNAs are involved in the regulation of oncogenic signaling networks between tumor cells and stroma is likely to add a needed additional layer of complexity to the tumor network, consisting of intercellular communication. More importantly, microRNA/EV signaling may provide an additional therapeutic target for this deadly disease.

The Ewing Sarcoma Protein (EWS) Binds Directly to the Proximal Elements of the Macrophage-Specific Promoter of the CSF-1 Receptor (csf1r) Gene

Many macrophage-specific promoters lack classical transcriptional start site elements such as TATA boxes and Sp1 sites. One example is the CSF-1 receptor (CSF-1R, CD115, c-fms), which is used as a model of the transcriptional regulation of macrophage genes. To understand the molecular basis of start site recognition in this gene, we identified cellular proteins binding specifically to the transcriptional start site (TSS) region. The mouse and human csf1r TSS were identified using cap analysis gene expression (CAGE) data. Conserved elements flanking the TSS cluster were analyzed using EMSAs to identify discrete DNA-binding factors in primary bone marrow macrophages as candidate transcriptional regulators. Two complexes were identified that bind in a highly sequence-specific manner to the mouse and human TSS proximal region and also to high-affinity sites recognized by myeloid zinc finger protein 1 (Mzf1). The murine proteins were purified by DNA affinity isolation from the RAW264.7 macrophage cell line and identified by mass spectrometry as EWS and FUS/TLS, closely related DNA and RNA-binding proteins. Chromatin immunoprecipitation experiments in bone marrow macrophages confirmed that EWS, but not FUS/TLS, was present in vivo on the CSF-1R proximal promoter in unstimulated primary macrophages. Transfection assays suggest that EWS does not act as a conventional transcriptional activator or repressor. We hypothesize that EWS contributes to start site recognition in TATA-less mammalian promoters.