Interstitial flow differentially increases patient-derived glioblastoma stem cell invasion via CXCR4, CXCL12, and CD44-mediated mechanisms.

Glioblastoma (GBM) prognosis remains dismal due in part to the invasiveness of GBM cells. Interstitial fluid flow (IFF) has been shown to increase invasion of glioma cells in vitro through the CXCR4 receptor interacting with autologous, pericellular gradients of CXCL12 (autologous chemotaxis) or through the CD44 receptor interactions with the extracellular matrix (hyaluronan-mediated mechanotransduction). These mechanisms have not been examined together and thus we hypothesized that both mechanisms contribute to invasion in populations of cancer cells. Therefore, we examined IFF-stimulated CXCR4-, CXCL12-, and CD44-dependent invasion in patient-derived glioblastoma stem cells (GSCs). Using our 3D in vitro assay and correlative in vivo studies we demonstrated GSC lines show increased invasion with flow. This flow-stimulated invasion was reduced by blockade of CXCR4, CXCL12, and/or CD44, revealing that GSC invasion may be mediated simultaneously by both mechanisms. Characterization of CXCR4+, CXCL12+, and CD44+ populations in four GSC lines revealed different percentages of protein positive subpopulations for each line. We developed an agent-based model to identify the contributions of each subpopulation to flow-stimulated invasion and validated the model through comparisons with experimental blocking studies. Clinically relevant radiation therapy increased flow-stimulated invasion in one GSC line. Our agent-based model predicted that IFF-stimulated invasion is driven primarily by CXCR4+CXCL12+ populations, and, indeed our irradiated cells had an increase in this subpopulation. Together, these data indicate that different mechanisms govern the flow response across GSCs, but that within a single patient, there are subpopulations of GSCs that respond to flow via either CD44- or CXCR4-CXCL12 mechanisms.

A miR-297/hypoxia/DGK-α axis regulating glioblastoma survival.

BACKGROUND: Despite advances in the treatment of the most aggressive form of brain tumor, glioblastoma, patient prognosis remains disappointing. This failure in treatment has been attributed to dysregulated oncogenic pathways, as observed in other tumors. We and others have suggested the use of microRNAs (miRs) as therapeutic tools able to target multiple pathways in glioblastoma. METHODS: This work features PCR quantification of miRs and transient transfection of many glioblastoma cell lines with miRs, followed by cell number analysis, trypan blue staining, alamarBlue assay of cell viability, caspase-3/-7 activity assay, immunoblot of cleaved poly(ADP-ribose) polymerase and fluorescence activated cell sorting and imaging of apoptotic nuclei, cell invasion assays, MRIs of glioblastoma xenografts in mice using transiently transfected cells as well as posttumor treatment with lentiviral vector encoding miR-297, and analysis of miR-297 target diacylglycerol kinase (DGK)-α including immunoblot, 3'UTR luciferase activity, and rescue with DGK-α overexpression. Cell counts and DGK-α immunoblot were also analyzed in the context of hypoxia and with overexpression of heterogeneous ribonucleoprotein L (hnRNPL). RESULTS: We identified miR-297 as a highly cytotoxic microRNA in glioblastoma, with minimal cytotoxicity to normal astrocytes. miR-297 overexpression reduced in vitro invasiveness and in vivo tumor formation. DGK-α is shown to be a miR-297 target with a critical role in miR-297 toxicity. In addition, hypoxia and its mediator hnRNPL upregulated DGK-α and buffered the cytotoxic effects of miR-297. CONCLUSION: This work shows miR-297 as a novel and physiologic regulator of cancer cell survival, largely through targeting of DGK-α, and also indicates that hypoxia ameliorates miR-297 toxicity to cancer cells.

Diacylglycerol kinase α is a critical signaling node and novel therapeutic target in glioblastoma and other cancers.

Although diacylglycerol kinase α (DGKα) has been linked to several signaling pathways related to cancer cell biology, it has been neglected as a target for cancer therapy. The attenuation of DGKα activity via DGKα-targeting siRNA and small-molecule inhibitors R59022 and R59949 induced caspase-mediated apoptosis in glioblastoma cells and in other cancers, but lacked toxicity in noncancerous cells. We determined that mTOR and hypoxia-inducible factor-1α (HIF-1α) are key targets of DGKα inhibition, in addition to its regulation of other oncogenes. DGKα regulates mTOR transcription via a unique pathway involving cyclic AMP. Finally, we showed the efficacy of DGKα inhibition with short hairpin RNA or a small-molecule agent in glioblastoma and melanoma xenograft treatment models, with growth delay and decreased vascularity. This study establishes DGKα as a central signaling hub and a promising therapeutic target in the treatment of cancer.

Frizzled receptors signal through G proteins.

Frizzled receptors have long been thought to couple to G proteins but biochemical evidence supporting such an interaction has been lacking. Here we expressed mammalian Wnt-Frizzled fusion proteins in Saccharomyces cerevisiae and tested the receptors' ability to activate the yeast mitogen-activated protein kinase (MAPK) pathway via heterotrimeric G proteins. Our results show that Frizzled receptors can interact with Gαi, Gαq, and Gαs proteins, thus confirming that Frizzled functions as a G protein coupled receptor (GPCR). However, the activity level of Frizzled-mediated G protein signaling was much lower than that of a typical GPCR and, surprisingly, was highest when coupled to Gαs. The Frizzled/Gαs interaction was further established in vivo as Drosophila expressing a loss-of-function Gαs allele rescued the photoreceptor differentiation phenotype of Frizzled mutant flies. Together, these data point to an important role for Frizzled as a nontraditional GPCR that preferentially couples to Gαs heterotrimeric G proteins.

Alpha-secretase inhibition reduces human glioblastoma stem cell growth in vitro and in vivo by inhibiting Notch.

The Notch pathway is dysregulated and a potential target in glioblastoma multiforme (GBM). Currently available Notch inhibitors block γ-secretase, which is necessary for Notch processing. However, Notch is first cleaved by α-secretase outside the plasma membrane, via a disintegrin and metalloproteinase-10 and -17. In this work, we used a potent α-secretase inhibitor (ASI) to test inhibition of glioblastoma growth and inhibition of Notch and of both novel and known Notch targets. Featured in this study are luciferase reporter assays and immunoblot, microarray analysis, chromatin immunoprecipitation (ChIP), quantitative real-time PCR, cell number assay, bromodeoxyuridine incorporation, plasmid rescue, orthotopic xenograft model, and local delivery of treatment with convection-enhanced delivery using nanoparticles, as well as survival, MRI, and ex vivo luciferase assay. A CBF1-luciferase reporter assay as well as an immunoblot of endogenous Notch revealed Notch inhibition by the ASI. Microarray analysis, quantitative real-time PCR, and ChIP of ASI and γ-secretase inhibitor (GSI) treatment of GBM cells identified known Notch pathway targets, as well as novel Notch targets, including YKL-40 and leukemia inhibitory factor. Finally, we found that local nanoparticle delivery of ASIs but not GSIs increased survival time significantly in a GBM stem cell xenograft treatment model, and ASI treatment resulted in decreased tumor size and Notch activity. This work indicates α-secretase as an alternative to γ-secretase for inhibition of Notch in GBM and possibly other cancers as well, and it identifies novel Notch targets with biologic relevance and potential as biomarkers.

The ADP receptor P2RY12 regulates osteoclast function and pathologic bone remodeling.

The adenosine diphosphate (ADP) receptor P2RY12 (purinergic receptor P2Y, G protein coupled, 12) plays a critical role in platelet aggregation, and P2RY12 inhibitors are used clinically to prevent cardiac and cerebral thrombotic events. Extracellular ADP has also been shown to increase osteoclast (OC) activity, but the role of P2RY12 in OC biology is unknown. Here, we examined the role of mouse P2RY12 in OC function. Mice lacking P2ry12 had decreased OC activity and were partially protected from age-associated bone loss. P2ry12-/- OCs exhibited intact differentiation markers, but diminished resorptive function. Extracellular ADP enhanced OC adhesion and resorptive activity of WT, but not P2ry12-/-, OCs. In platelets, ADP stimulation of P2RY12 resulted in GTPase Ras-related protein (RAP1) activation and subsequent αIIbß3 integrin activation. Likewise, we found that ADP stimulation induced RAP1 activation in WT and integrin ß3 gene knockout (Itgb3-/-) OCs, but its effects were substantially blunted in P2ry12-/- OCs. In vivo, P2ry12-/- mice were partially protected from pathologic bone loss associated with serum transfer arthritis, tumor growth in bone, and ovariectomy-induced osteoporosis: all conditions associated with increased extracellular ADP. Finally, mice treated with the clinical inhibitor of P2RY12, clopidogrel, were protected from pathologic osteolysis. These results demonstrate that P2RY12 is the primary ADP receptor in OCs and suggest that P2RY12 inhibition is a potential therapeutic target for pathologic bone loss.

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.

The bisphosphonate zoledronic acid decreases tumor growth in bone in mice with defective osteoclasts.

Bisphosphonates (BPs), bone targeted drugs that disrupt osteoclast function, are routinely used to treat complications of bone metastasis. Studies in preclinical models of cancer have shown that BPs reduce skeletal tumor burden and increase survival. Similarly, we observed in the present study that administration of the Nitrogen-containing BP (N-BP), zoledronic acid (ZA) to osteolytic tumor-bearing Tax+ mice beginning at 6 months of age led to resolution of radiographic skeletal lesions. N-BPs inhibit farnesyl diphosphate (FPP) synthase, thereby inhibiting protein prenylation and causing cellular toxicity. We found that ZA decreased Tax+ tumor and B16 melanoma viability and caused the accumulation of unprenylated Rap1a proteins in vitro. However, it is presently unclear whether N-BPs exert anti-tumor effects in bone independent of inhibition of osteoclast (OC) function in vivo. Therefore, we evaluated the impact of treatment with ZA on B16 melanoma bone tumor burden in irradiated mice transplanted with splenic cells from src(-/-) mice, which have non-functioning OCs. OC-defective mice treated with ZA demonstrated a significant 88% decrease in tumor growth in bone compared to vehicle-treated OC-defective mice. These data support an osteoclast-independent role for N-BP therapy in bone metastasis.

Interferon-gamma targets cancer cells and osteoclasts to prevent tumor-associated bone loss and bone metastases.

Interferon-gamma (IFN-gamma) has been shown to enhance anti-tumor immunity and inhibit the formation of bone-resorbing osteoclasts. We evaluated the role of IFN-gamma in bone metastases, tumor-associated bone destruction, and hypercalcemia in human T cell lymphotrophic virus type 1-Tax transgenic mice. Compared with Tax(+)IFN-gamma(+/+) mice, Tax(+)IFN-gamma(-/-) mice developed increased osteolytic bone lesions and soft tissue tumors, as well as increased osteoclast formation and activity. In vivo administration of IFN-gamma to tumor-bearing Tax(+)IFN-gamma(-/-) mice prevented new tumor development and resulted in decreased bromodeoxyuridine uptake by established tumors. In vitro, IFN-gamma directly decreased the viability of Tax(+) tumor cells through inhibition of proliferation, suppression of ERK phosphorylation, and induction of apoptosis and caspase 3 cleavage. IFN-gamma also inhibited macrophage colonystimulating factor-mediated proliferation and survival of osteoclast progenitors in vitro. Administration of IFN-gamma to C57BL/6 mice decreased Tax(+) tumor growth and prevented tumor-associated bone loss and hypercalcemia. In contrast, IFN-gamma treatment failed to protect IFN-gammaR1(-/-) mice from Tax(+) tumor-induced skeletal complications, despite decreasing tumor growth. These data demonstrate that IFN-gamma suppressed tumor-induced bone loss and hypercalcemia in Tax(+) mice by inhibiting both Tax(+) tumor cell growth and host-induced osteolysis. These data suggest a protective role for IFN-gamma in patients with bone metastases and hypercalcemia of malignancy.

APT102, a novel adpase, cooperates with aspirin to disrupt bone metastasis in mice.

Platelets contribute to the development of metastasis, the most common cause of mortality in cancer patients, but the precise role that anti-platelet drugs play in cancer treatment is not defined. Metastatic tumor cells can produce platelet alphaIIb beta3 activators, such as ADP and thromboxane A(2) (TXA(2)). Inhibitors of platelet beta3 integrins decrease bone metastases in mice but are associated with significant bleeding. We examined the role of a novel soluble apyrase/ADPase, APT102, and an inhibitor of TXA(2) synthesis, acetylsalicylic acid (aspirin or ASA), in mouse models of experimental bone metastases. We found that treatment with ASA and APT102 in combination (ASA + APT102), but not either drug alone, significantly decreased breast cancer and melanoma bone metastases in mice with fewer bleeding complications than observed with alphaIIb beta3 inhibition. ASA + APT102 diminished tumor cell induced platelet aggregation but did not directly alter tumor cell viability. Notably, APT102 + ASA treatment did not affect initial tumor cell distribution and similar results were observed in beta3-/- mice. These results show that treatment with ASA + APT102 decreases bone metastases without significant bleeding complications. Anti-platelet drugs such as ASA + APT102 could be valuable experimental tools for studying the role of platelet activation in metastasis as well as a therapeutic option for the prevention of bone metastases.

Random mutagenesis of the complement factor 5a (C5a) receptor N terminus provides a structural constraint for C5a docking.

The N terminus of G protein-coupled receptors has been implicated in binding to peptide hormones. We have used random saturation mutagenesis to identify essential residues in the N terminus of the human complement factor 5a receptor (C5aR). In a library of N-terminal mutant C5aR molecules screened for activation by C5a, residues 24-30 of the C5aR showed a marked propensity to mutate to cysteine, most likely indicating that sulfhydryl groups at these positions are appropriately situated to form disulfide interactions with the unpaired Cys(27) of human C5a. This presumptive spatial constraint allowed the ligand to be computationally docked to the receptor to form a model of the C5a/C5aR interaction. When the N-terminal mutant C5aR library was rescreened with C5a C27R, a ligand incapable of disulfide interactions, no individual position in the N terminus was essential for receptor signaling. However, the region 19-29 was relatively highly conserved in the functional mutants, further demonstrating that this region of the C5aR makes a productive physiologic interaction with the C5a ligand.

C5a receptor oligomerization. II. Fluorescence resonance energy transfer studies of a human G protein-coupled receptor expressed in yeast.

Recent studies demonstrate that members of the superfamily of G protein-coupled receptors (GPCRs) form oligomers both in vitro and in vivo. The mechanisms by which GPCRs oligomerize and the roles of accessory proteins in this process are not well understood. We used disulfide-trapping experiments to show that C5a receptors, expressed in mammalian cells, reside in membranes as oligomers (Klco, J. M., Lassere, T. B., and Baranski, T. J. (2003) J. Biol. Chem. 278, 35345-35353). To begin to address how C5a receptors form oligomers, we now use fluorescence resonance energy transfer experiments on human C5a receptors expressed in the lower eukaryote Saccharomyces cerevisiae. C5a receptors tagged with variants of the green fluorescent protein display energy transfer in intact yeast, demonstrating that mammalian accessory proteins are not required for C5a receptor oligomerization. In both intact yeast cells and membrane preparations, agonist does not affect FRET efficiency, and little energy transfer is observed between the C5a receptor and a co-expressed yeast pheromone receptor (encoded by STE2), indicating that C5a receptor oligomerization is both receptor-specific and constitutive. FRET studies performed on fractionated membranes demonstrate similar levels of energy transfer between tagged C5a receptors in endoplasmic reticulum compared with plasma membrane, and urea washing of membranes has little effect on the extent of energy transfer. The oligomerization of C5a receptors expressed in yeast displays characteristics similar to those observed for other GPCRs studied in mammalian cells. This model system should prove useful for further studies to define mechanisms of oligomerization of mammalian GPCRs.