Glioblastomas display cellular hierarchies containing tumor-propagating glioblastoma stem cells (GSCs). STAT3 is a critical signaling node in GSC maintenance but molecular mechanisms underlying STAT3 activation in GSCs are poorly defined. Here we demonstrate that the bone marrow X-linked (BMX) nonreceptor tyrosine kinase activates STAT3 signaling to maintain self-renewal and tumorigenic potential of GSCs. BMX is differentially expressed in GSCs relative to nonstem cancer cells and neural progenitors. BMX knockdown potently inhibited STAT3 activation, expression of GSC transcription factors, and growth of GSC-derived intracranial tumors. Constitutively active STAT3 rescued the effects of BMX downregulation, supporting that BMX signals through STAT3 in GSCs. These data demonstrate that BMX represents a GSC therapeutic target and reinforces the importance of STAT3 signaling in stem-like cancer phenotypes.

Tumors contain hostile inflammatory signals generated by aberrant proliferation, necrosis, and hypoxia. These signals are sensed and acted upon acutely by the Toll-like receptors (TLRs) to halt proliferation and activate an immune response. Despite the presence of TLR ligands within the microenvironment, tumors progress, and the mechanisms that permit this growth remain largely unknown. We report that self-renewing cancer stem cells (CSCs) in glioblastoma have low TLR4 expression that allows them to survive by disregarding inflammatory signals. Non-CSCs express high levels of TLR4 and respond to ligands. TLR4 signaling suppresses CSC properties by reducing retinoblastoma binding protein 5 (RBBP5), which is elevated in CSCs. RBBP5 activates core stem cell transcription factors, is necessary and sufficient for self-renewal, and is suppressed by TLR4 overexpression in CSCs. Our findings provide a mechanism through which CSCs persist in hostile environments because of an inability to respond to inflammatory signals.

Leukemia encompasses several hematological malignancies with shared phenotypes that include rapid proliferation, abnormal leukocyte self-renewal, and subsequent disruption of normal hematopoiesis. While communication between leukemia cells and the surrounding stroma supports tumor survival and expansion, the mechanisms underlying direct leukemia cell-cell communication and its contribution to tumor growth are undefined. Gap junctions are specialized intercellular connections composed of connexin proteins that allow free diffusion of small molecules and ions directly between the cytoplasm of adjacent cells. To characterize homotypic leukemia cell communication, we employed in vitro models for both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) and measured gap junction function through dye transfer assays. Additionally, clinically relevant gap junction inhibitors, carbenoxolone (CBX) and 1-octanol, were utilized to uncouple the communicative capability of leukemia cells. Furthermore, a qRT-PCR screen revealed several connexins with higher expression in leukemia cells compared with normal hematopoietic stem cells. Cx25 was identified as a promising adjuvant therapeutic target, and Cx25 but not Cx43 reduction via RNA interference reduced intercellular communication and sensitized cells to chemotherapy. Taken together, our data demonstrate the presence of homotypic communication in leukemia through a Cx25-dependent gap junction mechanism that can be exploited for the development of anti-leukemia therapies.

Colon cancer is a deadly disease affecting millions of people worldwide. Current treatment challenges include management of disease burden as well as improvements in detection and targeting of tumor cells. To identify disease state-specific surface antigen signatures, we combined fluorescent cell barcoding with high-throughput flow cytometric profiling of primary and metastatic colon cancer lines (SW480, SW620, and HCT116). Our multiplexed technique offers improvements over conventional methods by permitting the simultaneous and rapid screening of cancer cells with reduced effort and cost. The method uses a protein-level analysis with commercially available antibodies on live cells with intact epitopes to detect potential tumor-specific targets that can be further investigated for their clinical utility. Multiplexed antibody arrays can easily be applied to other tumor types or pathologies for discovery-based approaches to target identification.

Although vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) is traditionally regarded as an endothelial cell protein, evidence suggests that VEGFRs may be expressed by cancer cells. Glioblastoma multiforme (GBM) is a lethal cancer characterized by florid vascularization and aberrantly elevated VEGF. Antiangiogenic therapy with the humanized VEGF antibody bevacizumab reduces GBM tumor growth; however, the clinical benefits are transient and invariably followed by tumor recurrence. In this study, we show that VEGFR2 is preferentially expressed on the cell surface of the CD133(+) human glioma stem-like cells (GSCs), whose viability, self-renewal, and tumorigenicity rely, at least in part, on signaling through the VEGF-VEGFR2-Neuropilin-1 (NRP1) axis. We find that the limited impact of bevacizumab-mediated VEGF blockage may reflect ongoing autocrine signaling through VEGF-VEGFR2-NRP1, which is associated with VEGFR2-NRP1 recycling and a pool of active VEGFR2 within a cytosolic compartment of a subset of human GBM cells. Whereas bevacizumab failed to inhibit prosurvival effects of VEGFR2-mediated signaling, GSC viability under unperturbed or radiation-evoked stress conditions was attenuated by direct inhibition of VEGFR2 tyrosine kinase activity and/or shRNA-mediated knockdown of VEGFR2 or NRP1. We propose that direct inhibition of VEGFR2 kinase may block the highly dynamic VEGF-VEGFR2-NRP1 pathway and inspire a GBM treatment strategy to complement the currently prevalent ligand neutralization approach.

Accumulating evidence suggests that cancer cells with stem cell-like phenotypes drive disease progression and therapeutic resistance in glioblastoma (GBM). NOTCH regulates self-renewal and resistance to chemoradiotherapy in GBM stem cells. However, NOTCH-targeted γ-secretase inhibitors (GSIs) exhibited limited efficacy in GBM patients. We found that farnesyltransferase inhibitors (FTIs) significantly improved sensitivity to GSIs. This combination showed significant antineoplastic and radiosensitizing activities in GBM stem cells, whereas non-stem GBM cells were resistant. These combinatorial effects were mediated, at least partially, through inhibition of AKT and cell-cycle progression. Using subcutaneous and orthotopic GBM models, we showed that the combination of FTIs and GSIs, but not either agent alone, significantly reduced tumor growth. With concurrent radiation, this combination induced a durable response in a subset of orthotopic tumors. These findings collectively suggest that the combination of FTIs and GSIs is a promising therapeutic strategy for GBM through selectively targeting the cancer stem cell subpopulation.

The transmembrane protein, STRA6, functions as a vitamin A transporter and a cytokine receptor when activated by vitamin A-bound serum retinol binding protein 4 (RBP4). STRA6 activation transduces a JAK2-STAT3 signaling cascade and promotes tumorigenesis in a xenograft mouse model of colon cancer. We show here that RBP4 and STRA6 expression is associated with poor oncologic prognosis. Downregulating STRA6 or RBP4 in colon cancer cells decreased the fraction of cancer stem cells and their sphere and tumor initiation frequency. Furthermore, we show that high-fat diet (HFD) increases LGR5 expression and promotes tumor growth in a xenograft model independent of obesity. HFD increased STRA6 levels, and downregulation of STRA6 delays and impairs tumor initiation, tumor growth, and expression of stemness markers. Together, these data demonstrate a key role of STRA6 and RBP4 in the maintenance of colon cancer self-renewal and that this pathway is an important link through which consumption of HFD contributes to colon carcinogenesis.

The median survival for patients with glioblastoma (GBM), the most common primary malignant brain tumor in adults, has remained approximately 1 year for more than 2 decades. Recent advances in the field have identified GBM as a sexually dimorphic disease. It is less prevalent in females and they have better survival compared to males. The molecular mechanism of this difference has not yet been established. Iron is essential for many biological processes supporting tumor growth and its regulation is impacted by sex. Therefore, we interrogated the expression of a key component of cellular iron regulation, the HFE (homeostatic iron regulatory) gene, on sexually dimorphic survival in GBM.

An important factor correlated with poor survival in glioblastoma (GBM) is the aberrant and persistent activation of STAT3, a critical transcription factor that regulates multiple genes with key roles in cell survival, proliferation, resistance to chemotherapy, and stem cell maintenance. The Interleukin-6 (IL6)-STAT3 signaling axis has been studied extensively in inflammation and cancer. However, it is not completely understood how high levels of activated STAT3 are sustained in tumors. Previously, we identified a novel mechanism of biphasic activation of STAT3 in response to gp130-linked cytokines, including IL6, in which activation of STAT3 is prolonged by circumventing the negative regulatory mechanisms induced by its initial activationTo target prolonged STAT3 activation, we used the small molecule inhibitor bazedoxifene (BZA), which blocks formation of the IL6 receptor-gp130 complex. Glioma stem-like cells (GSCs) are more tumorigenic and more resistant to therapy. STAT3 is a key driver of the expression of stem cell transcription factors, making it a therapeutically important target in GBM. We show that treating GSCs with BZA decreases their self-renewal capacity and the expression of GSC markers in vitro. Additionally, BZA crosses the blood-brain barrier and confers a survival advantage in an orthotopic syngeneic mouse model of GBM. Although IL6-STAT3 signaling is important for GSC survival, a therapeutic agent that inhibits this pathway without toxicity has yet to be identified. Our findings reveal a mechanism of sustained STAT3 signaling in GBM and reveal its role in GSC maintenance, and we identify BZA as a novel candidate for treating GBM.

By including oligonucleotide-labeled antibodies into high-throughput single-cell RNA-sequencing protocols, combined transcript and protein expression data can be acquired on the single-cell level. Here, we describe a protocol for the combined analysis of over 40 proteins and 400 genes on over 104 cells using the nano-well based Rhapsody platform. We also include a workflow for sample multiplexing, which uniquely identifies the initial source of cells (such as tissue type or donor) in the downstream analysis after upstream pooling. For complete information on the use and execution of this protocol, please refer to Mair et al. (2020).

Glioblastoma (GBM) displays marked cellular and metabolic heterogeneity that varies among cellular microenvironments within a tumor. Metabolic targeting has long been advocated as a therapy against many tumors including GBM, but how lipid metabolism is altered to suit different microenvironmental conditions and whether cancer stem cells (CSCs) have altered lipid metabolism are outstanding questions in the field. We interrogated gene expression in separate microenvironments of GBM organoid models that mimic the transition between nutrient-rich and nutrient-poor pseudopalisading/perinecrotic tumor zones using spatial-capture RNA-sequencing. We revealed a striking difference in lipid processing gene expression and total lipid content between diverse cell populations from the same patient, with lipid enrichment in hypoxic organoid cores and also in perinecrotic and pseudopalisading regions of primary patient tumors. This was accompanied by regionally restricted upregulation of hypoxia-inducible lipid droplet-associated (HILPDA) gene expression in organoid cores and pseudopalisading regions of clinical GBM specimens, but not lower-grade brain tumors. CSCs have low lipid droplet accumulation compared to non-CSCs in organoid models and xenograft tumors, and prospectively sorted lipid-low GBM cells are functionally enriched for stem cell activity. Targeted lipidomic analysis of multiple patient-derived models revealed a significant shift in lipid metabolism between GBM CSCs and non-CSCs, suggesting that lipid levels may not be simply a product of the microenvironment but also may be a reflection of cellular state. CSCs had decreased levels of major classes of neutral lipids compared to non-CSCs, but had significantly increased polyunsaturated fatty acid production due to high fatty acid desaturase (FADS1/2) expression which was essential to maintain CSC viability and self-renewal. Our data demonstrate spatially and hierarchically distinct lipid metabolism phenotypes occur clinically in the majority of patients, can be recapitulated in laboratory models, and may represent therapeutic targets for GBM.

Migrating cell collectives are key to embryonic development but also contribute to invasion and metastasis of a variety of cancers. Cell collectives can invade deep into tissues, leading to tumor progression and resistance to therapies. Collective cell invasion is also observed in the lethal brain tumor glioblastoma (GBM), which infiltrates the surrounding brain parenchyma leading to tumor growth and poor patient outcomes. Drosophila border cells, which migrate as a small cell cluster in the developing ovary, are a well-studied and genetically accessible model used to identify general mechanisms that control collective cell migration within native tissue environments. Most cell collectives remain cohesive through a variety of cell-cell adhesion proteins during their migration through tissues and organs. In this study, we first identified cell adhesion, cell matrix, cell junction, and associated regulatory genes that are expressed in human brain tumors. We performed RNAi knockdown of the Drosophila orthologs in border cells to evaluate if migration and/or cohesion of the cluster was impaired. From this screen, we identified eight adhesion-related genes that disrupted border cell collective migration upon RNAi knockdown. Bioinformatics analyses further demonstrated that subsets of the orthologous genes were elevated in the margin and invasive edge of human GBM patient tumors. These data together show that conserved cell adhesion and adhesion regulatory proteins with potential roles in tumor invasion also modulate collective cell migration. This dual screening approach for adhesion genes linked to GBM and border cell migration thus may reveal conserved mechanisms that drive collective tumor cell invasion.

Synthetic lethality is a genetic interaction that results in cell death when two genetic deficiencies co-occur but not when either deficiency occurs alone, which can be co-opted for cancer therapeutics. Pairs of paralog genes are among the most straightforward potential synthetic-lethal interactions by virtue of their redundant functions. Here, we demonstrate a paralog-based synthetic lethality by targeting vaccinia-related kinase 1 (VRK1) in glioblastoma (GBM) deficient of VRK2, which is silenced by promoter methylation in approximately two thirds of GBM. Genetic knockdown of VRK1 in VRK2-null or VRK2-methylated cells resulted in decreased activity of the downstream substrate barrier to autointegration factor (BAF), a regulator of post-mitotic nuclear envelope formation. Reduced BAF activity following VRK1 knockdown caused nuclear lobulation, blebbing, and micronucleation, which subsequently resulted in G2-M arrest and DNA damage. The VRK1-VRK2 synthetic-lethal interaction was dependent on VRK1 kinase activity and was rescued by ectopic expression of VRK2. In VRK2-methylated GBM cell line-derived xenograft and patient-derived xenograft models, knockdown of VRK1 led to robust tumor growth inhibition. These results indicate that inhibiting VRK1 kinase activity could be a viable therapeutic strategy in VRK2-methylated GBM.

The ketogenic diet (KD) is hypothesized to impact tumor progression by altering tumor metabolism. In this study, we assessed the impact of an unrestricted KD on epithelial ovarian cancer (EOC) tumor growth, gene expression, and metabolite concentration in a mouse model. ID8 EOC cells, which were syngeneic with C57Bl/6J mouse strain and transfected with luciferase (ID8-luc), were injectedand monitored for tumor development. Female mice were fed either a strict KD, a high fat/low carbohydrate (HF/LC) diet, or a low fat/high carbohydrate (LF/HC) diet (n = 10 mice per group) ad libitum. EOC tumor growth was monitored weekly, and tumor burden was determined based on luciferase fluorescence (photons/second). At the endpoint (42 days), tumors were collected and processed for RNA sequencing. Plasma and tumor metabolites were evaluated using LC-MS. The KD-fed mice exhibited a statistically significant increase in tumor progression in comparison to the HF/LC- and LF/HC-fed groups (9.1 vs. 2.0 vs. 3.1-fold, respectively, p < 0.001). The EOC tumors of the KD-fed mice exhibited significant enrichment of the peroxisome proliferator-activated receptor (PPAR) signaling and fatty acid metabolism pathways based on the RNA sequencing analysis when compared to the LF/HC- and HF/LC-fed mice. Thus, unrestricted KD diet enhanced tumor progression in our mouse EOC model. KD was associated with the upregulation of fatty acid metabolism and regulation pathways, as well as enrichment of fatty acid and glutamine metabolites.

The actin-binding protein Kelch-like 1 (KLHL1) can modulate voltage-gated calcium channels in vitro. KLHL1 interacts with actin and with the pore-forming subunits of Cav2.1 and CaV3.2 calcium channels, resulting in up-regulation of P/Q and T-type current density. Here we tested whether endogenous KLHL1 modulates voltage gated calcium currents in cultured hippocampal neurons by down-regulating the expression of KLHL1 via adenoviral delivery of shRNA targeted against KLHL1 (shKLHL1). Control adenoviruses did not affect any of the neuronal properties measured, yet down-regulation of KLHL1 resulted in HVA current densities ~68% smaller and LVA current densities 44% smaller than uninfected controls, with a concomitant reduction in α(1A) and α(1H) protein levels. Biophysical analysis and western blot experiments suggest Ca(V)3.1 and 3.3 currents are also present in shKLHL1-infected neurons. Synapsin I levels, miniature postsynaptic current frequency, and excitatory and inhibitory synapse number were reduced in KLHL1 knockdown. This study corroborates the physiological role of KLHL1 as a calcium channel modulator and demonstrates a novel, presynaptic role.

Neural induction of human pluripotent stem cells often yields heterogeneous cell populations that can hamper quantitative and comparative analyses. There is a need for improved differentiation and enrichment procedures that generate highly pure populations of neural stem cells (NSC), glia and neurons. One way to address this problem is to identify cell-surface signatures that enable the isolation of these cell types from heterogeneous cell populations by fluorescence activated cell sorting (FACS).

Gap-junction-mediated cell-cell communication enables tumor cells to synchronize complex processes. We previously found that glioblastoma cancer stem cells (CSCs) express higher levels of the gap junction protein Cx46 compared to non-stem tumor cells (non-CSCs) and that this was necessary and sufficient for CSC maintenance. To understand the mechanism underlying this requirement, we use point mutants to disrupt specific functions of Cx46 and find that Cx46-mediated gap-junction coupling is critical for CSCs. To develop a Cx46 targeting strategy, we screen a clinically relevant small molecule library and identify clofazimine as an inhibitor of Cx46-specific cell-cell communication. Clofazimine attenuates proliferation, self-renewal, and tumor growth and synergizes with temozolomide to induce apoptosis. Although clofazimine does not cross the blood-brain barrier, the combination of clofazimine derivatives optimized for brain penetrance with standard-of-care therapies may target glioblastoma CSCs. Furthermore, these results demonstrate the importance of targeting cell-cell communication as an anti-cancer therapy.

Glioblastoma (GBM) is the most prevalent primary malignant brain tumor and is associated with extensive tumor cell infiltration into the adjacent brain parenchyma. However, there are limited targeted therapies that address this disease hallmark. While the invasive capacity of self-renewing cancer stem cells (CSCs) and their non-CSC progeny has been investigated, the mode(s) of migration used by CSCs during invasion is currently unknown. Here we used time-lapse microscopy to evaluate the migratory behavior of CSCs, with a focus on identifying key regulators of migration. A head-to-head migration assay demonstrated that CSCs are more invasive than non-CSCs. Time-lapse live cell imaging further revealed that GBM patient-derived CSC models either migrate in a collective manner or in a single cell fashion. To uncover conserved molecular regulators responsible for collective cell invasion, we utilized the genetically tractable Drosophila border cell collective migration model. Candidates for functional studies were generated using results from a targeted Drosophila genetic screen followed by gene expression analysis of the human homologs in GBM tumors and associated GBM patient prognosis. This strategy identified the highly conserved small GTPase, Rap1a, as a potential regulator of cell invasion. Alteration of Rap1a activity impaired the forward progress of Drosophila border cells during development. Rap1a expression was elevated in GBM and associated with higher tumor grade. Functionally, the levels of activated Rap1a impacted CSC migration speed out of spheres onto extracellular matrix. The data presented here demonstrate that CSCs are more invasive than non-CSCs, are capable of both collective and single cell migration, and express conserved genes that are required for migration and invasion. Using this integrated approach, we identified a new role for Rap1a in the migration of GBM CSCs.

Glioblastomas are among the most lethal cancers; however, recent advances in survival have increased the need for better prognostic markers. microRNAs (miRNAs) hold great prognostic potential being deregulated in glioblastomas and highly stable in stored tissue specimens. Moreover, miRNAs control multiple genes representing an additional level of gene regulation possibly more prognostically powerful than a single gene. The aim of the study was to identify a novel miRNA signature with the ability to separate patients into prognostic subgroups. Samples from 40 glioblastoma patients were included retrospectively; patients were comparable on all clinical aspects except overall survival enabling patients to be categorized as short-term or long-term survivors based on median survival. A miRNome screening was employed, and a prognostic profile was developed using leave-one-out cross-validation. We found that expression patterns of miRNAs; particularly the four miRNAs: hsa-miR-107_st, hsa-miR-548x_st, hsa-miR-3125_st and hsa-miR-331-3p_st could determine short- and long-term survival with a predicted accuracy of 78%. Heatmap dendrograms dichotomized glioblastomas into prognostic subgroups with a significant association to survival in univariate (HR 8.50; 95% CI 3.06-23.62; p<0.001) and multivariate analysis (HR 9.84; 95% CI 2.93-33.06; p<0.001). Similar tendency was seen in The Cancer Genome Atlas (TCGA) using a 2-miRNA signature of miR-107 and miR-331 (miR sum score), which were the only miRNAs available in TCGA. In TCGA, patients with O6-methylguanine-DNA-methyltransferase (MGMT) unmethylated tumors and low miR sum score had the shortest survival. Adjusting for age and MGMT status, low miR sum score was associated with a poorer prognosis (HR 0.66; 95% CI 0.45-0.97; p = 0.033). A Kyoto Encyclopedia of Genes and Genomes analysis predicted the identified miRNAs to regulate genes involved in cell cycle regulation and survival. In conclusion, the biology of miRNAs is complex, but the identified 4-miRNA expression pattern could comprise promising biomarkers in glioblastoma stratifying patients into short- and long-term survivors.

Glioblastoma (GBM) is the most common primary malignant brain tumor. Sex has been shown to be an important prognostic factor for GBM. The purpose of this study was to develop and independently validate sex-specific nomograms for estimation of individualized GBM survival probabilities using data from 2 independent NRG Oncology clinical trials.