Jakub Godlewski

microRNA-7 Inhibits the Epidermal Growth Factor Receptor and the Akt Pathway and Is Down-regulated in Glioblastoma

microRNAs are noncoding RNAs inhibiting expression of numerous target genes, and a few have been shown to act as oncogenes or tumor suppressors. We show that microRNA-7 (miR-7) is a potential tumor suppressor in glioblastoma targeting critical cancer pathways. miR-7 potently suppressed epidermal growth factor receptor expression, and furthermore it independently inhibited the Akt pathway via targeting upstream regulators. miR-7 expression was down-regulated in glioblastoma versus surrounding brain, with a mechanism involving impaired processing. Importantly, transfection with miR-7 decreased viability and invasiveness of primary glioblastoma lines. This study establishes miR-7 as a regulator of major cancer pathways and suggests that it has therapeutic potential for glioblastoma.

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.

The neuronal microRNA miR-326 acts in a feedback loop with Notch and has therapeutic potential against brain tumors

Little is known of microRNA interactions with cellular pathways. Few reports have associated microRNAs with the Notch pathway, which plays key roles in nervous system development and in brain tumors. We previously implicated the Notch pathway in gliomas, the most common and aggressive brain tumors. While investigating Notch mediators, we noted microRNA-326 was upregulated following Notch-1 knockdown. This neuronally expressed microRNA was not only suppressed by Notch but also inhibited Notch proteins and activity, indicating a feedback loop. MicroRNA-326 was downregulated in gliomas via decreased expression of its host gene. Transfection of microRNA-326 into both established and stem cell-like glioma lines was cytotoxic, and rescue was obtained with Notch restoration. Furthermore, miR-326 transfection reduced glioma cell tumorigenicity in vivo. Additionally, we found microRNA-326 partially mediated the toxic effects of Notch knockdown. This work demonstrates a microRNA-326/Notch axis, shedding light on the biology of Notch and suggesting microRNA-326 delivery as a therapy.

Micro-RNA dysregulation in multiple sclerosis favours pro-inflammatory T-cell-mediated autoimmunity

Pro-inflammatory T cells mediate autoimmune demyelination in multiple sclerosis. However, the factors driving their development and multiple sclerosis susceptibility are incompletely understood. We investigated how micro-RNAs, newly described as post-transcriptional regulators of gene expression, contribute to pathogenic T-cell differentiation in multiple sclerosis. miR-128 and miR-27b were increased in naïve and miR-340 in memory CD4(+) T cells from patients with multiple sclerosis, inhibiting Th2 cell development and favouring pro-inflammatory Th1 responses. These effects were mediated by direct suppression of B lymphoma Mo-MLV insertion region 1 homolog (BMI1) and interleukin-4 (IL4) expression, resulting in decreased GATA3 levels, and a Th2 to Th1 cytokine shift. Gain-of-function experiments with these micro-RNAs enhanced the encephalitogenic potential of myelin-specific T cells in experimental autoimmune encephalomyelitis. In addition, treatment of multiple sclerosis patient T cells with oligonucleotide micro-RNA inhibitors led to the restoration of Th2 responses. These data illustrate the biological significance and therapeutic potential of these micro-RNAs in regulating T-cell phenotypes in multiple sclerosis.

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.

MicroRNAs and glioblastoma; the stem cell connection

Recent data draw close parallels between cancer, including glial brain tumors, and the biology of stem and progenitor cells. At the same time, it has become clear that one of the major roles that microRNAs play is in the regulation of stem cell biology, differentiation, and cell ‘identity’. For example, microRNAs have been increasingly implicated in the regulation of neural differentiation. Interestingly, initial studies in the incurable brain tumor glioblastoma multiforme strongly suggest that microRNAs involved in neural development play a role in this disease. This encourages the idea that certain miRs allow continued tumor growth through the suppression of differentiation and the maintenance of the stem cell-like properties of tumor cells. These concepts will be explored in this article.

Lithium inhibits invasion of glioma cells; possible involvement of glycogen synthase kinase-3

Therapies targeting glioma cells that diffusely infiltrate normal brain are highly sought after. Our aim was to identify novel approaches to this problem using glioma spheroid migration assays. Lithium, a currently approved drug for the treatment of bipolar illnesses, has not been previously examined in the context of glioma migration. We found that lithium treatment potently blocked glioma cell migration in spheroid, wound-healing, and brain slice assays. The effects observed were dose dependent and reversible, and worked using every glioma cell line tested. In addition, there was little effect on cell viability at lithium concentrations that inhibit migration, showing that this is a specific effect. Lithium treatment was associated with a marked change in cell morphology, with cells retracting the long extensions at their leading edge. Examination of known targets of lithium showed that inositol monophosphatase inhibition had no effect on glioma migration, whereas inhibition of glycogen synthase kinase-3 (GSK-3) did. This suggested that the effects of lithium on glioma cell migration could possibly be mediated through GSK-3. Specific pharmacologic GSK-3 inhibitors and siRNA knockdown of GSK-3alpha or GSK-3beta isoforms both reduced cell motility. These data outline previously unidentified pathways and inhibitors that may be useful for the development of novel anti-invasive therapeutics for the treatment of brain tumors.

Oncogenic effects of miR-10b in glioblastoma stem cells

MicroRNAs and cancer stem cells have emerged as critical players in glioblastoma, one of the deadliest human cancers. In this study, we investigated the expression and function of microRNA-10b in glioblastoma cells and stem cells. An analysis of The Cancer Genome Atlas data revealed a correlation between high miR-10b levels and poor prognosis in glioblastoma patients. We measured the levels of miR-10b and found that it is upregulated in human glioblastoma tissues, glioblastoma cell and stem cell lines as compared to normal human tissues or astrocytes. Inhibition of miR-10b with a specific antagomir inhibited the proliferation of glioblastoma established and stem cell lines. Inhibition of miR-10b strongly reduced cell invasion and migration in glioblastoma cell and stem cell lines while overexpression of miR-10b induced cell migration and invasion. We also investigated several predicted targets of miR-10b but could not verify any of them experimentally. Additionally, miR-10b inhibition significantly decreased the in vivo growth of stem cell-derived orthotopic GBM xenografts. Altogether, our findings confirm the oncogenic effects of miR-10b in GBM cells and show for the first time a role of this microRNA in GBM stem cells. Targeting miR-10b might therefore inhibit glioblastoma stem cells, which are thought to be at the origin of glioblastoma and to contribute its recurrence and resistance to therapy.

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.