Yiai Tong

Prominin 1 marks intestinal stem cells that are susceptible to neoplastic transformation

Cancer stem cells are remarkably similar to normal stem cells: both self-renew, are multipotent and express common surface markers, for example, prominin 1 (PROM1, also called CD133). What remains unclear is whether cancer stem cells are the direct progeny of mutated stem cells or more mature cells that reacquire stem cell properties during tumour formation. Answering this question will require knowledge of whether normal stem cells are susceptible to cancer-causing mutations; however, this has proved difficult to test because the identity of most adult tissue stem cells is not known. Here, using an inducible Cre, nuclear LacZ reporter allele knocked into the Prom1 locus (Prom1C-L), we show that Prom1 is expressed in a variety of developing and adult tissues. Lineage-tracing studies of adult Prom1+/C-L mice containing the Rosa26-YFP reporter allele showed that Prom1+ cells are located at the base of crypts in the small intestine, co-express Lgr5 (ref. 2), generate the entire intestinal epithelium, and are therefore the small intestinal stem cell. Prom1 was reported recently to mark cancer stem cells of human intestinal tumours that arise frequently as a consequence of aberrant wingless (Wnt) signalling. Activation of endogenous Wnt signalling in Prom1+/C-L mice containing a Cre-dependent mutant allele of β-catenin (Ctnnb1lox(ex3)) resulted in a gross disruption of crypt architecture and a disproportionate expansion of Prom1+ cells at the crypt base. Lineage tracing demonstrated that the progeny of these cells replaced the mucosa of the entire small intestine with neoplastic tissue that was characterized by focal high-grade intraepithelial neoplasia and crypt adenoma formation. Although all neoplastic cells arose from Prom1+ cells in these mice, only 7% of tumour cells retained Prom1 expression. Our data indicate that Prom1 marks stem cells in the adult small intestine that are susceptible to transformation into tumours retaining a fraction of mutant Prom1+ tumour cells.

Subtypes of medulloblastoma have distinct developmental origins.

Medulloblastoma encompasses a collection of clinically and molecularly diverse tumour subtypes that together comprise the most common malignant childhood brain tumour. These tumours are thought to arise within the cerebellum, with approximately 25% originating from granule neuron precursor cells (GNPCs) after aberrant activation of the Sonic Hedgehog pathway (hereafter, SHH subtype). The pathological processes that drive heterogeneity among the other medulloblastoma subtypes are not known, hindering the development of much needed new therapies. Here we provide evidence that a discrete subtype of medulloblastoma that contains activating mutations in the WNT pathway effector CTNNB1 (hereafter, WNT subtype) arises outside the cerebellum from cells of the dorsal brainstem. We found that genes marking human WNT-subtype medulloblastomas are more frequently expressed in the lower rhombic lip (LRL) and embryonic dorsal brainstem than in the upper rhombic lip (URL) and developing cerebellum. Magnetic resonance imaging (MRI) and intra-operative reports showed that human WNT-subtype tumours infiltrate the dorsal brainstem, whereas SHH-subtype tumours are located within the cerebellar hemispheres. Activating mutations in Ctnnb1 had little impact on progenitor cell populations in the cerebellum, but caused the abnormal accumulation of cells on the embryonic dorsal brainstem which included aberrantly proliferating Zic1+ precursor cells. These lesions persisted in all mutant adult mice; moreover, in 15% of cases in which Tp53 was concurrently deleted, they progressed to form medulloblastomas that recapitulated the anatomy and gene expression profiles of human WNT-subtype medulloblastoma. We provide the first evidence, to our knowledge, that subtypes of medulloblastoma have distinct cellular origins. Our data provide an explanation for the marked molecular and clinical differences between SHH- and WNT-subtype medulloblastomas and have profound implications for future research and treatment of this important childhood cancer.

Active medulloblastoma enhancers reveal subgroup-specific cellular origins

Medulloblastoma is a highly malignant paediatric brain tumour, often inflicting devastating consequences on the developing child. Genomic studies have revealed four distinct molecular subgroups with divergent biology and clinical behaviour. An understanding of the regulatory circuitry governing the transcriptional landscapes of medulloblastoma subgroups, and how this relates to their respective developmental origins, is lacking. Here, using H3K27ac and BRD4 chromatin immunoprecipitation followed by sequencing (ChIP-seq) coupled with tissue-matched DNA methylation and transcriptome data, we describe the active cis-regulatory landscape across 28 primary medulloblastoma specimens. Analysis of differentially regulated enhancers and super-enhancers reinforced inter-subgroup heterogeneity and revealed novel, clinically relevant insights into medulloblastoma biology. Computational reconstruction of core regulatory circuitry identified a master set of transcription factors, validated by ChIP-seq, that is responsible for subgroup divergence, and implicates candidate cells of origin for Group 4. Our integrated analysis of enhancer elements in a large series of primary tumour samples reveals insights into cis-regulatory architecture, unrecognized dependencies, and cellular origins.

Siah Regulation of Pard3A Controls Neuronal Cell Adhesion During Germinal Zone Exit

Exiting the Birthplace In the developing mammalian brain, new neurons are not always born where they are needed. In order for immature neurons of the mouse cerebellum to leave their birthplace in the germinal zone and find their functional niche in the brain, the neurons need to migrate. Famulski et al. (p. 1834, published online 25 November; see the Perspective by Métin and Luccardini) now show that ubiquitin-mediated protein degradation regulates development of specific cell adhesions that the neurons need in order to exit their birthplace en route to their final functional location. A ubiquitination cascade regulates formation of cell adhesions that immature neurons require in the developing mouse brain. The brain’s circuitry is established by directed migration and synaptogenesis of neurons during development. Although neurons mature and migrate in specific patterns, little is known about how neurons exit their germinal zone niche. We found that cerebellar granule neuron germinal zone exit is regulated by proteasomal degradation of Pard3A by the Seven in Absentia homolog (Siah) E3 ubiquitin ligase. Pard3A gain of function and Siah loss of function induce precocious radial migration. Time-lapse imaging using a probe to measure neuronal cell contact reveals that Pard3A promotes adhesive interactions needed for germinal zone exit by recruiting the epithelial tight junction adhesion molecule C to the neuronal cell surface. Our findings define a Siah-Pard3A signaling pathway that controls adhesion-dependent exit of neuronal progenitors or immature neurons from a germinal zone niche.

Cross-Species Genomics Identifies TAF12, NFYC, and RAD54L as Choroid Plexus Carcinoma Oncogenes

Choroid plexus carcinomas (CPCs) are poorly understood and frequently lethal brain tumors with few treatment options. Using a mouse model of the disease and a large cohort of human CPCs, we performed a cross-species, genome-wide search for oncogenes within syntenic regions of chromosome gain. TAF12, NFYC, and RAD54L co-located on human chromosome 1p32-35.3 and mouse chromosome 4qD1-D3 were identified as oncogenes that are gained in tumors in both species and required for disease initiation and progression. TAF12 and NFYC are transcription factors that regulate the epigenome, whereas RAD54L plays a central role in DNA repair. Our data identify a group of concurrently gained oncogenes that cooperate in the formation of CPC and reveal potential avenues for therapy.

Orthotopic models of pediatric brain tumors in zebrafish

High-throughput screens (HTS) of compound toxicity against cancer cells can identify thousands of potential new drug-leads. But only limited numbers of these compounds can progress to expensive and labor-intensive efficacy studies in mice, creating a ‘bottle neck’ in the drug development pipeline. Approaches that triage drug-leads for further study are greatly needed. Here we provide an intermediary platform between HTS and mice by adapting mouse models of pediatric brain tumors to grow as orthotopic xenografts in the brains of zebrafish. Freshly isolated mouse ependymoma, glioma and choroid plexus carcinoma cells expressing red fluorescence protein were conditioned to grow at 34 °C. Conditioned tumor cells were then transplanted orthotopically into the brains of zebrafish acclimatized to ambient temperatures of 34 °C. Live in vivo fluorescence imaging identified robust, quantifiable and reproducible brain tumor growth as well as spinal metastasis in zebrafish. All tumor xenografts in zebrafish retained the histological characteristics of the corresponding parent mouse tumor and efficiently recruited fish endothelial cells to form a tumor vasculature. Finally, by treating zebrafish harboring ERBB2-driven gliomas with an appropriate cytotoxic chemotherapy (5-fluorouracil) or tyrosine kinase inhibitor (erlotinib), we show that these models can effectively assess drug efficacy. Our data demonstrate, for the first time, that mouse brain tumors can grow orthotopically in fish and serve as a platform to study drug efficacy. As large cohorts of brain tumor-bearing zebrafish can be generated rapidly and inexpensively, these models may serve as a powerful tool to triage drug-leads from HTS for formal efficacy testing in mice.

Abstract 3448: Subtypes of medulloblastoma have distinct developmental origins

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Medulloblastoma encompasses a collection of clinically and molecularly diverse tumor subtypes that together comprise the most common malignant childhood brain tumor. These tumors are thought to arise within the cerebellum, with approximately 25% originating from granule neuron precursor cells (GNPCs) following aberrant activation of the Sonic Hedgehog pathway (hereafter, SHH-subtype). The pathological processes that drive heterogeneity among the other medulloblastoma subtypes are not known, hindering the development of much needed new therapies. Here, we provide evidence that a discrete subtype of medulloblastoma that contains activating mutations in the WNT pathway effector CTNNB1 (hereafter, WNT-subtype), arises outside the cerebellum from cells of the lower rhombic lip (LRL). We found that genes marking human WNT-subtype medulloblastomas are more frequently expressed in the LRL and embryonic dorsal brainstem than in the upper rhombic lip (URL) and developing cerebellum. Magnetic resonance imaging (MRI) and intra-operative reports showed that human WNT-subtype tumors infiltrate the dorsal brainstem, while SHH-subtype tumors are located within the cerebellar hemispheres. Activating mutations in Ctnnb1 had little impact on progenitor cell populations in the cerebellum, but caused an aberrant accumulation of proliferating precursor cells within the LRL. These lesions persisted in the dorsal brainstem of all mutant adult mice and in 15% of cases in which Tp53 was concurrently deleted, progressed to form medulloblastomas that modeled faithfully the anatomy and gene expression profiles of human WNT-subtype medulloblastoma. We provide the first evidence that subtypes of medulloblastoma have distinct cellular origins. Our data provide an explanation for the marked molecular and clinical differences between SHH and WNT-subtype medulloblastomas and have profound implications for future research and treatment of this important childhood cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3448. doi:10.1158/1538-7445.AM2011-3448

Abstract 4759: Integrated in vitro and in vivo screening of tumor and normal neural stem cells identifies potential new treatments of ependymoma

Ependymomas are rare brain tumors that are incurable unless completely excised. The low incidence of the disease and lack of pre-clinical models has limited research efforts to advance understanding of biology and treatment. Recently, we used interspecies genomics to match specific driver mutations with distinct types of mouse neural stem cell (NSC) to accurately model human ependymoma. Here we report the use of these models for high throughput drug screening (HTS). Stem-like mouse ependymoma cells (mEPCs), non-ependymoma mouse brain tumor cells (mBTCs) and control transduced NSCs (mNSCs) were cultured in neurosphere conditions adapted for use in an automated small molecule HTS. We first performed replicate primary screens of 7,579 agents drawn from a bioactive library, FDA approved drug library and kinase library. Primary screens were conducted in a single concentration format (10µM). The primary screen was highly reproducible and ROC analysis of primary screen data was used to assess predictive power of the screen (ROC AUC>0.89 (0.85-0.92 95% CI)). A total of 602 compounds representing diverse drug classes progressed from primary to secondary screening. These included full 10-point dose response assays that identified a total of 181 agents with activity in at least one cell population. In all, 2.3% of compounds displayed anti-mEPC activity and were enriched for anti-cancer drugs (Fishers Exact P=1.9 × 10-7: Bonferroni correction threshold, p=0.0016). Since our HTS strategy included non-ependymoma tumor cells and mNSCs, we were able to refine our classification of activity to define compounds more active against mEPCs than other cells (0.08%); equally active against mEPCs and mBTCs relative to mNSCs (0.04%); equally active against all four cell types (2.2%); more active against mBTCs relative to all other cells (0.2%); more active against mNSCs relative to tumor cells (0.8%); inactive against mEPCs relative to all other cells (0.1%); and inactive against all four cell types (96%). Interestingly, anticancer compounds displayed patterns of cell-selective activity that varied according to their mechanism of action with some drug classes appearing significantly more toxic to mNSCs than either mEPCs or mBTCs. These HTS studies identified treatments for ependymoma including drugs that were relatively non-toxic to normal NSCs. Five agents were selected for assessment of in vivo against the originating ependymoma mouse models. Using the xenogen system to monitor tumor growth and assessing animal survival, we identified FDA-approved agents with activity in ependymoma which have not previously been implicated in the disease, and may be translated directly into the clinic. In summary, this approach identified a number of potential new treatments with potent activity against ependymoma relative to normal NSCs, and could be used to develop effective therapies for other rare cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4759. doi:10.1158/1538-7445.AM2011-4759