Johanna M. Niers

Human Glioma Growth Is Controlled by MicroRNA-10b

MicroRNA (miRNA) expression profiling studies revealed a number of miRNAs dysregulated in the malignant brain tumor glioblastoma. Molecular functions of these miRNAs in gliomagenesis are mainly unknown. We show that inhibition of miR-10b, a miRNA not expressed in human brain and strongly upregulated in both low-grade and high-grade gliomas, reduces glioma cell growth by cell-cycle arrest and apoptosis. These cellular responses are mediated by augmented expression of the direct targets of miR-10b, including BCL2L11/Bim, TFAP2C/AP-2γ, CDKN1A/p21, and CDKN2A/p16, which normally protect cells from uncontrolled growth. Analysis of The Cancer Genome Atlas expression data set reveals a strong positive correlation between numerous genes sustaining cellular growth and miR-10b levels in human glioblastomas, while proapoptotic genes anticorrelate with the expression of miR-10b. Furthermore, survival of glioblastoma patients expressing high levels of miR-10 family members is significantly reduced in comparison to patients with low miR-10 levels, indicating that miR-10 may contribute to glioma growth in vivo. Finally, inhibition of miR-10b in a mouse model of human glioma results in significant reduction of tumor growth. Altogether, our experiments validate an important role of miR-10b in gliomagenesis, reveal a novel mechanism of miR-10b-mediated regulation, and suggest the possibility of its future use as a therapeutic target in gliomas.

In Silico Analysis of Kinase Expression Identifies WEE1 as a Gatekeeper against Mitotic Catastrophe in Glioblastoma

Kinases execute pivotal cellular functions and are therefore widely investigated as potential targets in anticancer treatment. Here we analyze the kinase gene expression profiles of various tumor types and reveal the wee1 kinase to be overexpressed in glioblastomas. We demonstrate that WEE1 is a major regulator of the G(2) checkpoint in glioblastoma cells. Inhibition of WEE1 by siRNA or small molecular compound in cells exposed to DNA damaging agents results in abrogation of the G(2) arrest, premature termination of DNA repair, and cell death. Importantly, we show that the small-molecule inhibitor of WEE1 sensitizes glioblastoma to ionizing radiation in vivo. Our results suggest that inhibition of WEE1 kinase holds potential as a therapeutic approach in treatment of glioblastoma.

Down-Regulation of miR-101 in Endothelial Cells Promotes Blood Vessel Formation through Reduced Repression of EZH2

Angiogenesis is a balanced process controlled by pro- and anti-angiogenic molecules of which the regulation is not fully understood. Besides classical gene regulation, miRNAs have emerged as post-transcriptional regulators of angiogenesis. Furthermore, epigenetic changes caused by histone-modifying enzymes were shown to modulate angiogenesis as well. However, a possible interplay between miRNAs and histone-modulating enzymes during angiogenesis has not been described. Here we show that VEGF-mediated down-regulation of miR-101 caused pro-angiogenic effects. We found that the pro-angiogenic effects are partly mediated through reduced repression by miR-101 of the histone-methyltransferase EZH2, a member of the Polycomb group family, thereby increasing methylation of histone H3 at lysine 27 and transcriptome alterations. In vitro, the sprouting and migratory properties of primary endothelial cell cultures were reduced by inhibiting EZH2 through up-regulation of miR-101, siRNA-mediated knockdown of EZH2, or treatment with 3-Deazaneplanocin-A (DZNep), a small molecule inhibitor of EZH2 methyltransferase activity. In addition, we found that systemic DZNep administration reduced the number of blood vessels in a subcutaneous glioblastoma mouse model, without showing adverse toxicities. Altogether, by identifying a pro-angiogenic VEGF/miR-101/EZH2 axis in endothelial cells we provide evidence for a functional link between growth factor-mediated signaling, post-transcriptional silencing, and histone-methylation in the angiogenesis process. Inhibition of EZH2 may prove therapeutic in diseases in which aberrant vascularization plays a role.

Gaussia luciferase variant for high-throughput functional screening applications.

Gaussia luciferase (Gluc) is a sensitive reporter for studying different biological processes such as gene expression, promoter activity, protein-protein interactions, signal transduction, as well as tumor cell growth and response to therapy. Since Gluc is naturally secreted, the kinetics of these processes can be monitored in real-time by measuring an aliquot of conditioned medium in culture or a few microliters of blood in vivo at different time points. Gluc catalyzes light emission with a short half-life which is unfavorable for certain applications. We isolated a Gluc mutant that catalyzes enhanced light stability in the presence of a detergent, in combination with high sensitivity, making it an attractive luciferase for high-throughput functional screening applications.

Effects of the Selective MPS1 Inhibitor MPS1-IN-3 on Glioblastoma Sensitivity to Antimitotic Drugs

BACKGROUND Glioblastomas exhibit a high level of chemotherapeutic resistance, including to the antimitotic agents vincristine and taxol. During the mitotic agent-induced arrest, glioblastoma cells are able to perform damage-control and self-repair to continue proliferation. Monopolar spindle 1 (MPS1/TTK) is a checkpoint kinase and a gatekeeper of the mitotic arrest. METHODS We used glioblastoma cells to determine the expression of MPS1 and to determine the effects of MPS1 inhibition on mitotic errors and cell viability in combination with vincristine and taxol. The effect of MPS1 inhibition was assessed in different orthotopic glioblastoma mouse models (n = 3-7 mice/group). MPS1 expression levels were examined in relation to patient survival. RESULTS Using publicly available gene expression data, we determined that MPS1 overexpression corresponds positively with tumor grade and negatively with patient survival (two-sided t test, P < .001). Patients with high MPS1 expression (n = 203) had a median and mean survival of 487 and 913 days (95% confidence intervals [CI] = 751 to 1075), respectively, and a 2-year survival rate of 35%, whereas patients with intermediate MPS1 expression (n = 140) had a median and mean survival of 858 and 1183 days (95% CI = 1177 to 1189), respectively, and a 2-year survival rate of 56%. We demonstrate that MPS1 inhibition by RNAi results in sensitization to antimitotic agents. We developed a selective small-molecule inhibitor of MPS1, MPS1-IN-3, which caused mitotic aberrancies in glioblastoma cells and, in combination with vincristine, induced mitotic checkpoint override, increased aneuploidy, and augmented cell death. MPS1-IN-3 sensitizes glioblastoma cells to vincristine in orthotopic mouse models (two-sided log-rank test, P < .01), resulting in prolonged survival without toxicity. CONCLUSIONS Our results collectively demonstrate that MPS1, a putative therapeutic target in glioblastoma, can be selectively inhibited by MPS1-IN-3 sensitizing glioblastoma cells to antimitotic drugs.

Real-time monitoring of nuclear factor kappaB activity in cultured cells and in animal models.

Nuclear factor κB (NF-κB) is a transcription factor that plays a major role in many human disorders, including immune diseases and cancer. We designed a reporter system based on NF-κB responsive promoter elements driving expression of the secreted Gaussia princeps luciferase (Gluc). We show that this bioluminescent reporter is a highly sensitive tool for noninvasive monitoring of the kinetics of NF-κB activation and inhibition over time, both in conditioned medium of cultured cells and in the blood and urine of animals. NF-κB activation was successfully monitored in real time in endothelial cells in response to tumor angiogenic signaling, as well as in monocytes in response to inflammation. Further, we demonstrated dual blood monitoring of both NF-κB activation during tumor development as correlated to tumor formation using the NF-κB Gluc reporter, as well as the secreted alkaline phosphatase reporter. This NF-κB reporter system provides a powerful tool for monitoring NF-κB activity in real time in vitro and in vivo.

Triple Bioluminescence Imaging for In Vivo Monitoring of Cellular Processes

Bioluminescence imaging (BLI) has shown to be crucial for monitoring in vivo biological processes. So far, only dual bioluminescence imaging using firefly (Fluc) and Renilla or Gaussia (Gluc) luciferase has been achieved due to the lack of availability of other efficiently expressed luciferases using different substrates. Here, we characterized a codon-optimized luciferase from Vargula hilgendorfii (Vluc) as a reporter for mammalian gene expression. We showed that Vluc can be multiplexed with Gluc and Fluc for sequential imaging of three distinct cellular phenomena in the same biological system using vargulin, coelenterazine, and D-luciferin substrates, respectively. We applied this triple imaging system to monitor the effect of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) delivered using an adeno-associated viral vector (AAV) on brain tumors in mice. Vluc imaging showed efficient sTRAIL gene delivery to the brain, while Fluc imaging revealed a robust antiglioma therapy. Further, nuclear factor-κB (NF-κB) activation in response to sTRAIL binding to glioma cells death receptors was monitored by Gluc imaging. This work is the first demonstration of trimodal in vivo bioluminescence imaging and will have a broad applicability in many different fields including immunology, oncology, virology, and neuroscience.

Single Reporter for Targeted Multimodal in Vivo Imaging

We have developed a multifaceted, highly specific reporter for multimodal in vivo imaging and applied it for detection of brain tumors. A metabolically biotinylated, membrane-bound form of Gaussia luciferase was synthesized, termed mbGluc-biotin. We engineered glioma cells to express this reporter and showed that brain tumor formation can be temporally imaged by bioluminescence following systemic administration of coelenterazine. Brain tumors expressing this reporter had high sensitivity for detection by magnetic resonance and fluorescence tomographic imaging upon injection of streptavidin conjugated to magnetic nanoparticles or fluorophore, respectively. Moreover, single photon emission computed tomography showed enhanced imaging of these tumors upon injection with streptavidin complexed to (111)In-DTPA-biotin. This work shows for the first time a single small reporter (∼40 kDa) which can be monitored with most available molecular imaging modalities and can be extended for single cell imaging using intravital microscopy, allowing real-time tracking of any cell expressing it in vivo.

Multimodal In Vivo Imaging and Blood Monitoring of Intrinsic and Extrinsic Apoptosis

Noninvasive detection and in vivo imaging of apoptosis plays a critical role in the development of therapeutics in many different fields including cancer. We have developed an apoptosis biosensor by fusing green fluorescent protein (GFP) to the N-terminus of the naturally secreted Gaussia luciferase separated by a caspase-3 cleavage peptide consisting of aspartic acid (D), glutamic acid (E), valine (V), and aspartic acid (D) or DEVD. We showed that this fusion is retained in the cytoplasm of cells in an inactive form. Upon apoptosis, the DEVD peptide is cleaved in response to caspase-3 activation, freeing ssGluc, which can now enter the secretory pathway where it is folded properly and is released from the cells and can be detected in the conditioned medium in culture or in blood of live animals ex vivo over time. Because Gluc is secreted from cells via conventional pathway through the endoplasmic reticulum (ER), Golgi and vesicles, we showed that the presence of Gluc in these compartments in response to apoptosis can be visualized in vivo using bioluminescence imaging. This reporter provides a valuable tool for imaging and real-time monitoring of apoptosis and is compatible with high-throughput functional screening application in cultured cells and animal models.

Lanatoside C sensitizes glioblastoma cells to tumor necrosis factor–related apoptosis-inducing ligand and induces an alternative cell death pathway

Human glioblastoma (GBM) cells are notorious for their resistance to apoptosis-inducing therapeutics. We have identified lanatoside C as a sensitizer of GBM cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death partly by upregulation of the death receptor 5. We show that lanatoside C sensitizes GBM cells to TRAIL-induced apoptosis in a GBM xenograft model in vivo. Lanatoside C on its own serves as a therapeutic agent against GBM by activating a caspase-independent cell death pathway. Cells treated with lanatoside C showed necrotic cell morphology with absence of caspase activation, low mitochondrial membrane potential, and early intracellular ATP depletion. In conclusion, lanatoside C sensitizes GBM cells to TRAIL-induced cell death and mitigates apoptosis resistance of glioblastoma cells by inducing an alternative cell death pathway. To our knowledge, this is one of the first examples of use of caspase-independent cell death inducers to trigger tumor regression in vivo. Activation of such mechanism may be a useful strategy to counter resistance of cancer cells to apoptosis.