Sean E. Lawler

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.

MicroRNAs in cancer: biomarkers, functions and therapy

The emergence of microRNAs has been one of the defining developments in cancer biology over the past decade, and the explosion of knowledge in this area has brought forward new diagnostic and therapeutic opportunities. The importance of microRNAs in cancer has been underlined by the identification of alterations in microRNA target binding sites and the microRNA processing machinery in tumor cells. Clinical trials utilizing microRNA profiling for patient prognosis and clinical response are now underway, and the first microRNA mimic entered the clinic for cancer therapy in 2013. In this article we review the potential applications of microRNAs for the clinical assessment of patient outcome in cancer, as well as in cancer monitoring and therapy.

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.

Depletion of Peripheral Macrophages and Brain Microglia Increases Brain Tumor Titers of Oncolytic Viruses

Clinical trials have proven oncolytic virotherapy to be safe but not effective. We have shown that oncolytic viruses (OV) injected into intracranial gliomas established in rodents are rapidly cleared, and this is associated with up-regulation of markers (CD68 and CD163) of cells of monocytic lineage (monocytes/microglia/macrophages). However, it is unclear whether these cells directly impede intratumoral persistence of OV through phagocytosis and whether they infiltrate the tumor from the blood or the brain parenchyma. To investigate this, we depleted phagocytes with clodronate liposomes (CL) in vivo through systemic delivery and ex vivo in brain slice models with gliomas. Interestingly, systemic CL depleted over 80% of peripheral CD163+ macrophages in animal spleen and peripheral blood, thereby decreasing intratumoral infiltration of these cells, but CD68+ cells were unchanged. Intratumoral viral titers increased 5-fold. In contrast, ex vivo CL depleted only CD68+ cells from brain slices, and intratumoral viral titers increased 10-fold. These data indicate that phagocytosis by both peripheral CD163+ and brain-resident CD68+ cells infiltrating tumor directly affects viral clearance from tumor. Thus, improved therapeutic efficacy may require modulation of these innate immune cells. In support of this new therapeutic paradigm, we observed intratumoral up-regulation of CD68+ and CD163+ cells following treatment with OV in a patient with glioblastoma.

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.

NK cells impede glioblastoma virotherapy through NKp30 and NKp46 natural cytotoxicity receptors

The role of the immune response to oncolytic Herpes simplex viral (oHSV) therapy for glioblastoma is controversial because it might enhance or inhibit efficacy. We found that within hours of oHSV infection of glioblastomas in mice, activated natural killer (NK) cells are recruited to the site of infection. This response substantially diminished the efficacy of glioblastoma virotherapy. oHSV-activated NK cells coordinated macrophage and microglia activation within tumors. In vitro, human NK cells preferentially lysed oHSV-infected human glioblastoma cell lines. This enhanced killing depended on the NK cell natural cytotoxicity receptors (NCRs) NKp30 and NKp46, whose ligands are upregulated in oHSV-infected glioblastoma cells. We found that HSV titers and oHSV efficacy are increased in Ncr1−/− mice and a Ncr1−/− NK cell adoptive transfer model of glioma, respectively. These results demonstrate that glioblastoma virotherapy is limited partially by an antiviral NK cell response involving specific NCRs, uncovering new potential targets to enhance cancer virotherapy.

Emerging functions of microRNAs in glioblastoma.

Distinct patterns of microRNA expression have been observed in glioblastomas. The functional significance of some of these microRNAs is beginning to emerge. This data indicates that microRNAs play roles in multiple hallmark biological characteristics of glioblastoma, including cell proliferation, invasion, glioma stem cell behavior, and angiogenesis. Research in this area is quickly gathering pace and is illuminating important aspects of the disease that may ultimately lead to novel therapeutic interventions, as well as diagnostic and prognostic tools for brain tumors.