Nicole McFarlane

Sonic hedgehog regulates Bmi1 in human medulloblastoma brain tumor-initiating cells

Bmi1 is a key stem cell regulatory gene implicated in the pathogenesis of many aggressive cancers, including medulloblastoma. Overexpression of Bmi1 promotes cell proliferation and is required for hedgehog (Hh) pathway-driven tumorigenesis. This study aimed to determine if Sonic hedgehog (Shh) modulates the key stem cell regulatory gene Bmi1 in childhood medulloblastoma brain tumor-initiating cells (BTICs). Although current literature suggests that there is a correlation between Shh pathway genes and Bmi1 expression, it is unclear whether there is indeed a direct regulatory mechanism. To address whether Shh induces expression of Bmi1, stem cell-enriched populations from medulloblastoma cell lines and primary samples were treated with Shh ligand and KAAD-cyclopamine (Shh antagonist). Our data indicate that Bmi1 expression positively correlates with increasing Shh ligand concentrations. Chromatin immunoprecipitation reveals that Gli1 preferentially binds to the Bmi1 promoter, and Bmi1 transcript levels are increased and decreased by Gli1 overexpression and downregulation, respectively. Knockdown experiments of Bmi1 in vitro and in vivo demonstrate that Hh signaling not only drives Bmi1 expression, but a feedback mechanism exists wherein downstream effectors of Bmi1 may, in turn, activate Hh pathway genes. These findings implicate Bmi1 and Hh as mutually indispensable pathways in medulloblastoma BTIC maintenance. Recent molecular characterization of medulloblastoma also reveals that Bmi1 is overexpressed across all subgroups of medulloblastoma, particularly in the most aggressive subtypes. Lastly, despite recent identification of BTIC markers, the molecular characterization of these cell populations remains unclear. In this work, we propose that the BTIC marker CD133 may segregate a cell population with a Hh-receptor phenotype, thus demonstrating a cell–cell interaction between the CD133+ Hh receptor cells and the CD133− Hh-secreting cells.

Bax forms an oligomer via separate, yet interdependent, surfaces

Interactions of Bcl-2 family proteins regulate permeability of the mitochondrial outer membrane and apoptosis. In particular, Bax forms an oligomer that permeabilizes the membrane. To map the interface of the Bax oligomer we used Triton X-100 as a membrane surrogate and performed site-specific photocross-linking. Bax-specific adducts were formed through photo-reactive probes at multiple sites that can be grouped into two surfaces. The first surface overlaps with the BH1–3 groove formed by Bcl-2 Homology motif 1, 2, and 3; the second surface is a rear pocket located on the opposite side of the protein from the BH1–3 groove. Further cross-linking experiments using Bax BH3 peptides and mutants demonstrated that the two surfaces interact with their counterparts in neighboring proteins to form two separated interfaces and that interaction at the BH1–3 groove primes the rear pocket for further interaction. Therefore, Bax oligomerization proceeds through a series of interactions that occur at separate, yet allosterically, coupled interfaces.

Medulloblastoma stem cells: where development and cancer cross pathways

Brain tumors are the leading cause of childhood cancer mortality, with medulloblastoma (MB) representing the most frequent malignant tumor. The recent molecular classification of MB has reconceptualized the heterogeneity that exists within pathological subtypes by giving context to the role of key developmental signaling pathways in MB pathogenesis. The identification of cancer stem cell (CSC) populations, termed brain tumor-initiating cells (BTICs), in MB has provided novel cellular targets for the study of these aberrantly activated signaling pathways, namely, Sonic hedgehog (Shh) and Wingless (Wnt), along with the identification of novel BTIC self-renewal pathways. In this review, we discuss recent evidence for the presence of a MB stem cell that drives tumorigenesis in this malignant childhood tumor. We focus on evidence from cerebellar development, the recent identification of BTICs, the presence of activated developmental signaling pathways in MB, the role of epigenetic stem cell regulatory mechanisms, and how these developmental and epigenetic pathways may be targeted for novel therapeutic options.

A Cancer Stem Cell Model for Studying Brain Metastases From Primary Lung Cancer

BACKGROUND Brain metastases are most common in adults with lung cancer, predicting uniformly poor patient outcome, with a median survival of only months. Despite their frequency and severity, very little is known about tumorigenesis in brain metastases. METHODS We applied previously developed primary solid tumor-initiating cell models to the study of brain metastases from the lung to evaluate the presence of a cancer stem cell population. Patient-derived brain metastases (n = 20) and the NCI-H1915 cell line were cultured as stem-enriching tumorspheres. We used in vitro limiting-dilution and sphere-forming assays, as well as intracranial human-mouse xenograft models. To determine genes overexpressed in brain metastasis tumorspheres, we performed comparative transcriptome analysis. All statistical analyses were two-sided. RESULTS Patient-derived brain metastasis tumorspheres had a mean sphere-forming capacity of 33 spheres/2000 cells (SD = 33.40) and median stem-cell frequency of 1/60 (range = 0-1/141), comparable to that of primary brain tumorspheres (P = .53 and P = .20, respectively). Brain metastases also expressed CD15 and CD133, markers suggestive of a stemlike population. Through intracranial xenotransplantation, brain metastasis tumorspheres were found to recapitulate the original patient tumor heterogeneity. We also identified several genes overexpressed in brain metastasis tumorspheres as statistically significant predictors of poor survival in primary lung cancer. CONCLUSIONS For the first time, we demonstrate the presence of a stemlike population in brain metastases from the lung. We also show that NCI-H1915 tumorspheres could be useful in studying self-renewal and tumor initiation in brain metastases. Our candidate genes may be essential to metastatic stem cell populations, where pathway interference may be able to transform a uniformly fatal disease into a more localized and treatable one.

FoxG1 Interacts with Bmi1 to Regulate Self‐Renewal and Tumorigenicity of Medulloblastoma Stem Cells

Brain tumors represent the leading cause of childhood cancer mortality, of which medulloblastoma (MB) is the most frequent malignant tumor. Recent studies have demonstrated the presence of several MB molecular subgroups, each distinct in terms of prognosis and predicted therapeutic response. Groups 1 and 2 are characterized by relatively good clinical outcomes and activation of the Wnt and Shh pathways, respectively. In contrast, groups 3 and 4 (“non‐Shh/Wnt MBs”) are distinguished by metastatic disease, poor patient outcome, and lack a molecular pathway phenotype. Current gene expression platforms have not detected brain tumor‐initiating cell (BTIC) self‐renewal genes in groups 3 and 4 MBs as BTICs typically comprise a minority of tumor cells and may therefore go undetected on bulk tumor analyses. Since increasing BTIC frequency has been associated with increasing tumor aggressiveness and poor patient outcome, we investigated the subgroup‐specific gene expression profile of candidate stem cell genes within 251 primary human MBs from four nonoverlapping MB transcriptional databases (Amsterdam, Memphis, Toronto, Boston) and 74 NanoString‐subgrouped MBs (Vancouver). We assessed the functional relevance of two genes, FoxG1 and Bmi1, which were significantly enriched in non‐Shh/Wnt MBs and showed these genes to mediate MB stem cell self‐renewal and tumor initiation in mice. We also identified their transcriptional regulation through reciprocal promoter occupancy in CD15+ MB stem cells. Our work demonstrates the application of stem cell data gathered from genomic platforms to guide functional BTIC assays, which may then be used to develop novel BTIC self‐renewal mechanisms amenable to therapeutic targeting. STEM Cells2013;31:1266–1277

Regulation of SR-BI-mediated selective lipid uptake in Chinese hamster ovary-derived cells by protein kinase signaling pathways

Scavenger receptor, class B, type I (SR-BI) mediates binding and internalization of a variety of lipoprotein and nonlipoprotein ligands, including HDL. Studies in genetically engineered mice revealed that SR-BI plays an important role in HDL reverse cholesterol transport and protection against atherosclerosis. Understanding how SR-BIs function is regulated may reveal new approaches to therapeutic intervention in atherosclerosis and heart disease. We utilized a model cell system to explore pathways involved in SR-BI-mediated lipid uptake from and signaling in response to distinct lipoprotein ligands: the physiological ligand, HDL, and a model ligand, acetyl LDL (AcLDL). In Chinese hamster ovary-derived cells, murine SR-BI (mSR-BI) mediates lipid uptake via distinct pathways that are dependent on the lipoprotein ligand. Furthermore, HDL and AcLDL activate distinct signaling pathways. Finally, mSR-BI-mediated selective lipid uptake versus endocytic uptake are differentially regulated by protein kinase signaling pathways. The protein kinase C (PKC) activator PMA and the phosphatidyl inositol 3-kinase inhibitor wortmannin increase the degree of mSR-BI-mediated selective lipid uptake, whereas a PKC inhibitor has the opposite effect. These data demonstrate that SR-BIs selective lipid uptake activity can be acutely regulated by intracellular signaling cascades, some of which can originate from HDL binding to murine SR-BI itself.

Radiation-induced apoptosis in mouse lymphocytes is modified by a complex dietary supplement: the effect of genotype and gender

This study examined whether radiation sensitivity measured by lymphocyte apoptosis could be ameliorated by a complex anti-oxidant/anti-ageing dietary supplement. We also examined lymphocytes from both genders of normal (Nr) mice as well as transgenic growth hormone (Tg) mice that express strongly elevated reactive oxygen species processes and a progeroid syndrome of accelerated ageing. We introduce Tg mice as a potentially valuable new model to study radiation sensitivity. Isolated lymphocytes from all experimental groups were exposed to gamma radiation and the time course of apoptosis was measured in vitro. Kinetics of radiation-induced apoptosis was similar among groups, which peaked at 8 h, but maximal levels differed significantly between groups. Nr male mice had 60% lower levels of radiation-induced apoptosis than Tg males, supporting our hypothesis that Tg mice would be radiation sensitive. The dietary supplement protected lymphocytes in male mice of both strains, with proportionally greater reductions in Tg mice. Lymphocytes from female mice (both Nr and Tg) were highly radiation resistant compared to males and the supplement provided no additional benefit at the doses used in this study. These results highlight that radiation-induced apoptosis is complex and is modified by genotype, dietary supplements and gender.

The inhibition of endocytosis affects HDL-lipid uptake mediated by the human scavenger receptor class B type I

The scavenger receptor SR-BI plays an important role in the hepatic clearance of HDL cholesterol and other lipids, driving reverse cholesterol transport and contributing to protection against atherosclerosis in mouse models. We characterized the role of endocytosis in lipid uptake from HDL, mediated by the human SR-BI, using a variety of approaches to inhibit endocytosis, including hypertonic shock, potassium or energy depletion and disassembly of the actin cytoskeleton. Our studies revealed that unlike mouse SR-BI, human SR-BI-mediated HDL-lipid uptake was reduced by inhibition of endocytosis. This was not dependent on the cytoplasmic C-terminus of SR-BI. Monitoring the uptake of both the protein and lipid components of HDL revealed that although overall lipid uptake was decreased, the degree of selective lipid uptake was increased. These data suggest that that endocytosis is a dynamic regulator of SR-BIs selective lipid uptake activity.

Culture and Isolation of Brain Tumor Initiating Cells

Brain tumors are typically composed of heterogeneous cells that exhibit distinct phenotypic characteristics and proliferative potentials. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. This unit describes protocols for the culture and isolation BTICs. We applied culture conditions and assays originally used for normal neural stem cells (NSCs) in vitro to a variety of brain tumors. Using fluorescence-activated cell sorting for the neural precursor cell surface marker CD133/CD15, BTICs can be isolated and studied prospectively. Isolation of BTICs from GBM bulk tumor will enable examination of dissimilar morphologies, self-renewal capacities, tumorigenicity, and therapeutic sensitivities. As cancer is also considered a disease of unregulated self-renewal and differentiation, an understanding of BTICs is fundamental to understanding tumor growth. Ultimately, it will lead to novel drug discovery approaches that strategically target the functionally relevant BTIC population.

Pyrvinium Targets CD133 in Human Glioblastoma Brain Tumor–Initiating Cells

Purpose: Clonal evolution of cancer may be regulated by determinants of stemness, specifically self-renewal, and current therapies have not considered how genetic perturbations or properties of stemness affect such functional processes. Glioblastoma-initiating cells (GICs), identified by expression of the cell surface marker CD133, are shown to be chemoradioresistant. In the current study, we sought to elucidate the functional role of CD133 in self-renewal and identify compounds that can specifically target this CD133+ treatment-refractory population. Experimental Design: Using gain/loss-of-function studies for CD133 we assessed the in vitro self-renewal and in vivo tumor formation capabilities of patient-derived glioblastoma cells. We generated a CD133 signature combined with an in silico screen to find compounds that target GICs. Self-renewal and proliferation assays on CD133-sorted samples were performed to identify the preferential action of hit compounds. In vivo efficacy of the lead compound pyrvinium was assessed in intracranial GIC xenografts and survival studies. Lastly, microarray analysis was performed on pyrvinium-treated GICs to discover core signaling events involved. Results: We discovered pyrvinium, a small-molecule inhibitor of GIC self-renewal in vitro and in vivo, in part through inhibition of Wnt/β-catenin signaling and other essential stem cell regulatory pathways. We provide a therapeutically tractable strategy to target self-renewing, chemoradioresistant, and functionally important CD133+ stem cells that drive glioblastoma relapse and mortality. Conclusions: Our study provides an integrated approach for the eradication of clonal populations responsible for cancer progression, and may apply to other aggressive and heterogeneous cancers. Clin Cancer Res; 21(23); 5324–37. ©2015 AACR.