Tamra Werbowetski-Ogilvie

Isolation of a natural inhibitor of human malignant glial cell invasion : Inter α-trypsin inhibitor heavy chain 2

Malignant central nervous system (CNS) tumors, such as glioblastoma multiforme, invade the brain and disrupt normal tissue architecture, making complete surgical removal virtually impossible. Here, we have developed and optimized a purification strategy to isolate and identify natural inhibitors of glioma cell invasion in a three-dimensional collagen type I matrix. Inter alpha-trypsin inhibitor heavy chain 2 (ITI H2) was identified from the most inhibitory fractions and its presence was confirmed both as a single protein and in a bikunin-bound form. Stable overexpression in U251 glioma cells validated ITI H2s strong inhibition of human glioma cell invasion together with significant inhibition of cell proliferation and promotion of cell-cell adhesion. Analysis of primary human brain tumors showed significantly higher levels of ITI H2 in normal brain and low-grade tumors compared with high-grade gliomas, indicating an inverse correlation with malignancy. The phosphatidylinositol 3-kinase/Akt signaling cascade seemed to be one of the pathways involved in the effect of ITI H2 on U251 cells. These findings suggest that reduction of ITI H2 expression correlates with brain tumor progression and that targeting factors responsible for its loss or restoring the ITI supply exogenously may serve as potential therapeutic strategies for a variety of CNS tumors.

Differential cellular responses induced by dorsomorphin and LDN-193189 in chemotherapy-sensitive and chemotherapy-resistant human epithelial ovarian cancer cells

Inherent or acquired drug resistance is a major contributor to epithelial ovarian cancer (EOC) mortality. Novel drugs or drug combinations that produce EOC cell death or resensitize drug resistant cells to standard chemotherapy may improve patient treatment. After conducting drug tolerability studies for the multikinase inhibitors dorsomorphin (DM) and it is structural analogue LDN‐193189 (LDN), these drugs were tested in a mouse intraperitoneal xenograft model of EOC. DM significantly increased survival, whereas LDN showed a trend toward increased survival. In vitro experiments using cisplatin (CP)‐resistant EOC cell lines, A2780‐cp or SKOV3, we determined that pretreatment or cotreatment with DM or LDN resensitized cells to the killing effect of CP or carboplatin (CB). DM was capable of blocking EOC cell cycle and migration, whereas LDN produced a less pronounced effect on cell cycle and no effect on migration. Subsequent analyses using primary human EOC cell samples or additional established EOC cells lines showed that DM or LDN induced a dose‐dependent autophagic or cell death response, respectively. DM induced a characteristic morphological change with the appearance of numerous LC3B‐containing acidic vacuoles and an increase in LC3BII levels. This was coincident with a decrease in cell growth and the altered cell cycle consistent with DM‐induced cytostasis. By contrast, LDN produced a caspase 3‐independent, reactive oxygen species‐dependent cell death. Overall, DM and LDN possess drug characteristics suitable for adjuvant agents used to treat chemotherapy‐sensitive and ‐resistant EOC.

Characterization of novel biomarkers in selecting for subtype specific medulloblastoma phenotypes.

Major research efforts have focused on defining cell surface marker profiles for characterization and selection of brain tumor stem/progenitor cells. Medulloblastoma is the most common primary malignant pediatric brain cancer and consists of 4 molecular subgroups: WNT, SHH, Group 3 and Group 4. Given the heterogeneity within and between medulloblastoma variants, surface marker profiles may be subtype-specific. Here, we employed a high throughput flow cytometry screen to identify differentially expressed cell surface markers in self-renewing vs. non-self-renewing SHH medulloblastoma cells. The top 25 markers were reduced to 4, CD271/p75NTR/NGFR, CD106/VCAM1, EGFR and CD171/NCAM-L1, by evaluating transcript levels in SHH tumors relative to samples representing the other variants. However, only CD271/p75NTR/NGFR and CD171/NCAM-L1 maintain differential expression between variants at the protein level. Functional characterization of CD271, a low affinity neurotrophin receptor, in cell lines and primary cultures suggested that CD271 selects for lower self-renewing progenitors or stem cells. Moreover, CD271 levels were negatively correlated with expression of SHH pathway genes. Our study reveals a novel role for CD271 in SHH medulloblastoma and suggests that targeting CD271 pathways could lead to the design of more selective therapies that lessen the broad impact of current treatments on developing nervous systems.

OTX2 exhibits cell-context-dependent effects on cellular and molecular properties of human embryonic neural precursors and medulloblastoma cells

ABSTRACT Medulloblastoma (MB) is the most common malignant primary pediatric brain tumor and is currently divided into four subtypes based on different genomic alterations, gene expression profiles and response to treatment: WNT, Sonic Hedgehog (SHH), Group 3 and Group 4. This extensive heterogeneity has made it difficult to assess the functional relevance of genes to malignant progression. For example, expression of the transcription factor Orthodenticle homeobox2 (OTX2) is frequently dysregulated in multiple MB variants; however, its role may be subtype specific. We recently demonstrated that neural precursors derived from transformed human embryonic stem cells (trans-hENs), but not their normal counterparts (hENs), resemble Groups 3 and 4 MB in vitro and in vivo. Here, we tested the utility of this model system as a means of dissecting the role of OTX2 in MB using gain- and loss-of-function studies in hENs and trans-hENs, respectively. Parallel experiments with MB cells revealed that OTX2 exerts inhibitory effects on hEN and SHH MB cells by regulating growth, self-renewal and migration in vitro and tumor growth in vivo. This was accompanied by decreased expression of pluripotent genes, such as SOX2, and was supported by overexpression of SOX2 in OTX2+ SHH MB and hENs that resulted in significant rescue of self-renewal and cell migration. By contrast, OTX2 is oncogenic and promotes self-renewal of trans-hENs and Groups 3 and 4 MB independent of pluripotent gene expression. Our results demonstrate a novel role for OTX2 in self-renewal and migration of hENs and MB cells and reveal a cell-context-dependent link between OTX2 and pluripotent genes. Our study underscores the value of human embryonic stem cell derivatives as alternatives to cell lines and heterogeneous patient samples for investigating the contribution of key developmental regulators to MB progression. Summary: Human embryonic stem cell neural derivatives can be used to model the molecular and cellular properties of medulloblastoma.

Animal Models of Cancer Stem Cells: What are They Really Telling Us?

The cancer stem cell hypothesis suggests that a subpopulation of tumor cells exhibits stem cell properties such as self-renewal capacity and multilineage differentiation. The modern day cancer stem cell model emerged following initial work in leukemia and subsequent identification of putative cancer stem cell populations in a variety of solid tumors. These studies have been largely based on transplantation assays in immunodeficient mice and have revolutionized how we view tumor cell heterogeneity. However, recent studies with both genetically engineered and more highly immunocompromised animal models have challenged the notion that cancer stem cells are a rare subpopulation. Issues with the cell surface markers used to enrich for cancer stem cells and the hierarchical organization within tumors have also come to light and remain unresolved. As we continue to optimize in vivo models and understand microenvironmental influences, our view of cancer stem cells within heterogeneous tumors will likely become even more complex.

Characterization of a novel OTX2‐driven stem cell program in Group 3 and Group 4 medulloblastoma

Medulloblastoma (MB) is the most common malignant primary pediatric brain cancer. Among the most aggressive subtypes, Group 3 and Group 4 originate from stem/progenitor cells, frequently metastasize, and often display the worst prognosis, yet we know the least about the molecular mechanisms driving their progression. Here, we show that the transcription factor orthodenticle homeobox 2 (OTX2) promotes self‐renewal while inhibiting differentiation in vitro and increases tumor initiation from MB stem/progenitor cells in vivo. To determine how OTX2 contributes to these processes, we employed complementary bioinformatic approaches to characterize the OTX2 regulatory network and identified novel relationships between OTX2 and genes associated with neuronal differentiation and axon guidance signaling in Group 3 and Group 4 MB stem/progenitor cells. In particular, OTX2 levels were negatively correlated with semaphorin (SEMA) signaling, as expression of 9 SEMA pathway genes is upregulated following OTX2 knockdown with some being potential direct OTX2 targets. Importantly, this negative correlation was also observed in patient samples, with lower expression of SEMA4D associated with poor outcome specifically in Group 4 tumors. Functional proof‐of‐principle studies demonstrated that increased levels of select SEMA pathway genes are associated with decreased self‐renewal and growth in vitro and in vivo and that RHO signaling, known to mediate the effects of SEMA genes, is contributing to the OTX2 KD phenotype. Our study provides mechanistic insight into the networks controlled by OTX2 in MB stem/progenitor cells and reveals novel roles for axon guidance genes and their downstream effectors as putative tumor suppressors in MB.

CD271+ Cells Are Diagnostic and Prognostic and Exhibit Elevated MAPK Activity in SHH Medulloblastoma

The extensive heterogeneity both between and within the medulloblastoma subgroups underscores a critical need for variant-specific biomarkers and therapeutic strategies. We previously identified a role for the CD271/p75 neurotrophin receptor (p75NTR) in regulating stem/progenitor cells in the SHH medulloblastoma subgroup. Here, we demonstrate the utility of CD271 as a novel diagnostic and prognostic marker for SHH medulloblastoma using IHC analysis and transcriptome data across 763 primary tumors. RNA sequencing of CD271+ and CD271- cells revealed molecularly distinct, coexisting cellular subsets, both in vitro and in vivo MAPK/ERK signaling was upregulated in the CD271+ population, and inhibiting this pathway reduced endogenous CD271 levels, stem/progenitor cell proliferation, and cell survival as well as cell migration in vitro Treatment with the MEK inhibitor selumetinib extended survival and reduced CD271 levels in vivo, whereas, treatment with vismodegib, a well-known smoothened (SMO) inhibitor currently in clinical trials for the treatment of recurrent SHH medulloblastoma, had no significant effect in our models. Our study demonstrates the clinical utility of CD271 as both a diagnostic and prognostic tool for SHH medulloblastoma tumors and reveals a novel role for MEK inhibitors in targeting CD271+ SHH medulloblastoma cells.Significance: This study identifies CD271 as a specific and novel biomarker of SHH-type medulloblastoma and that targeting CD271+ cells through MEK inhibition represents a novel therapeutic strategy for the treatment of SHH medulloblastoma. Cancer Res; 78(16); 4745-59. ©2018 AACR.

Primary Pediatric Brain Tumors of the Posterior Fossa Part II: A Comprehensive Overview of Medulloblastoma

Medulloblastoma (MB) is the most common malignant primary brain tumor in children and is currently classified into five distinct molecular subtypes (WNT, SHH-TP53 wild type, SHH-TP53 mutant, Group 3, and Group 4), based on genomic alterations, gene expression profiles, responses to treatment, and developmental cell of origin. The standard treatment for MB consists of surgical resection followed by radiation therapy and chemotherapy. However, current treatments do not take into account the extensive heterogeneity between and within MB subtypes. Cancer stem cells also play an important role in treatment failure and recurrence in MB, adding an additional layer of complexity in the form of cellular heterogeneity. This chapter will focus on the clinical presentation of MB, current treatment options, and histological classifications with a more detailed description of the current molecular subtypes, followed by exploration of cellular heterogeneity in the molecular era. Further dissection of tumor heterogeneity and identification of subtype-specific biomarkers will be crucial in the development of novel diagnostic markers and targeted therapies for these highly aggressive pediatric brain tumors.

Primary Pediatric Brain Tumors of the Posterior Fossa: Part I

In pediatric neuro-oncology practice, cerebellar tumors are often referred to as infratentorial tumors or tumors of the posterior fossa (a differential diagnosis is provided in Table 1). This anatomic region also contains the pons and medulla, which along with the midbrain comprise the brainstem. In Part I of this comprehensive review, three important pediatric brain tumors usually localized to the cerebellum are discussed (and summarized in Table 2): atypical teratoid/rhabdoid tumors (ATRTs), pilocytic astrocytomas, and ependymomas. In the companion chapter (Part II), an integrated clinical and molecular overview of medulloblastoma follows. These tumors have been selected, in part, due to their clinical significance as well as recent advances in their molecular genetics and pathological classification. For these entities and others, the histopathologic, cytogenetic, and molecular factors have been integrated into the updated fourth edition of the World Health Organization (WHO) Classification of Tumors of the Central Nervous System (Louis et al., WHO classification of tumours of the central nervous system, Revised 4th edn. IARC, Lyon, 2016a; Louis et al., Acta Neuropathol 131:803–820, 2016b).

Novel glycolipid agents for killing cisplatin-resistant human epithelial ovarian cancer cells

BackgroundChemotherapy resistance is one of the major factors contributing to mortality from human epithelial ovarian cancer (EOC). Identifying drugs that can effectively kill chemotherapy-resistant EOC cells would be a major advance in reducing mortality. Glycosylated antitumour ether lipids (GAELs) are synthetic glycolipids that are cytotoxic to a wide range of cancer cells. They appear to induce cancer cell death in an apoptosis-independent manner.MethodsHerein, the effectiveness of two GAELs, GLN and MO-101, in killing chemotherapy-sensitive and –resistant EOC cells lines and primary cell samples was tested using monolayer, non-adherent aggregate, and non-adherent spheroid cultures.ResultsOur results show that EOC cells exhibit a differential sensitivity to the GAELs. Strikingly, both GAELs are capable of inducing EOC cell death in chemotherapy-sensitive and –resistant cells grown as monolayer or non-adherent cultures. Mechanistic studies provide evidence that apoptotic-cell death (caspase activation) contributes to, but is not completely responsible for, GAEL-induced cell killing in the A2780-cp EOC cell line, but not primary EOC cell samples.ConclusionsStudies using primary EOC cell samples supports previously published work showing a GAEL-induced caspase-independent mechanism of death. GAELs hold promise for development as novel compounds to combat EOC mortality due to chemotherapy resistance.