Yanjiao Li

A restricted cell population propagates glioblastoma growth after chemotherapy

Glioblastoma multiforme is the most common primary malignant brain tumour, with a median survival of about one year. This poor prognosis is due to therapeutic resistance and tumour recurrence after surgical removal. Precisely how recurrence occurs is unknown. Using a genetically engineered mouse model of glioma, here we identify a subset of endogenous tumour cells that are the source of new tumour cells after the drug temozolomide (TMZ) is administered to transiently arrest tumour growth. A nestin-ΔTK-IRES-GFP (Nes-ΔTK-GFP) transgene that labels quiescent subventricular zone adult neural stem cells also labels a subset of endogenous glioma tumour cells. On arrest of tumour cell proliferation with TMZ, pulse-chase experiments demonstrate a tumour re-growth cell hierarchy originating with the Nes-ΔTK-GFP transgene subpopulation. Ablation of the GFP+ cells with chronic ganciclovir administration significantly arrested tumour growth, and combined TMZ and ganciclovir treatment impeded tumour development. Thus, a relatively quiescent subset of endogenous glioma cells, with properties similar to those proposed for cancer stem cells, is responsible for sustaining long-term tumour growth through the production of transient populations of highly proliferative cells.

Pten Regulates Neuronal Arborization and Social Interaction in Mice

CNS deletion of Pten in the mouse has revealed its roles in controlling cell size and number, thus providing compelling etiology for macrocephaly and Lhermitte-Duclos disease. PTEN mutations in individuals with autism spectrum disorders (ASD) have also been reported, although a causal link between PTEN and ASD remains unclear. In the present study, we deleted Pten in limited differentiated neuronal populations in the cerebral cortex and hippocampus of mice. Resulting mutant mice showed abnormal social interaction and exaggerated responses to sensory stimuli. We observed macrocephaly and neuronal hypertrophy, including hypertrophic and ectopic dendrites and axonal tracts with increased synapses. This abnormal morphology was associated with activation of the Akt/mTor/S6k pathway and inactivation of Gsk3beta. Thus, our data suggest that abnormal activation of the PI3K/AKT pathway in specific neuronal populations can underlie macrocephaly and behavioral abnormalities reminiscent of certain features of human ASD.

Malignant Astrocytomas Originate from Neural Stem/Progenitor Cells in a Somatic Tumor Suppressor Mouse Model

Malignant astrocytomas are infiltrative and incurable brain tumors. Despite profound therapeutic implications, the identity of the cell (or cells) of origin has not been rigorously determined. We previously reported mouse models based on conditional inactivation of the human astrocytoma-relevant tumor suppressors p53, Nf1, and Pten, wherein through somatic loss of heterozygosity, mutant mice develop tumors with 100% penetrance. In the present study, we show that tumor suppressor inactivation in neural stem/progenitor cells is both necessary and sufficient to induce astrocytoma formation. We demonstrate in vivo that transformed cells and their progeny undergo infiltration and multilineage differentiation during tumorigenesis. Tumor suppressor heterozygous neural stem/progenitor cultures from presymptomatic mice show aberrant growth advantage and altered differentiation, thus identifying a pretumorigenic cell population.

Pten Haploinsufficiency Accelerates Formation of High-Grade Astrocytomas

We previously reported that central nervous system (CNS) inactivation of Nf1 and p53 tumor suppressor genes in mice results in the development of low-grade to high-grade progressive astrocytomas. When the tumors achieve high grade, they are frequently accompanied by Akt activation, reminiscent of the frequent association of PTEN mutations in human high-grade glioma. In the present study, we introduced CNS heterozygosity of Pten into the Nf1/p53 astrocytoma model. Resulting mice had accelerated morbidity, shortened survival, and full penetrance of high-grade astrocytomas. Haploinsufficiency of Pten accelerated formation of grade 3 astrocytomas, whereas loss of Pten heterozygosity and Akt activation coincided with progression into grade 4 tumors. These data suggest that successive loss of each Pten allele may contribute to de novo formation of high-grade astrocytoma and progression into glioblastoma, respectively, thus providing insight into the etiology of primary glioblastoma. The presence of ectopically migrating neural stem/progenitor lineage cells in presymptomatic Pten-deficient mutant brains supports the notion that these tumors may arise from stem/progenitor cells.

CXCR4/CXCL12 mediate autocrine cell- cycle progression in NF1-associated malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas that arise in connective tissue surrounding peripheral nerves. They occur sporadically in a subset of patients with neurofibromatosis type 1 (NF1). MPNSTs are highly aggressive, therapeutically resistant, and typically fatal. Using comparative transcriptome analysis, we identified CXCR4, a G-protein-coupled receptor, as highly expressed in mouse models of NF1-deficient MPNSTs, but not in nontransformed precursor cells. The chemokine receptor CXCR4 and its ligand, CXCL12, promote MPNST growth by stimulating cyclin D1 expression and cell-cycle progression through PI3-kinase (PI3K) and β-catenin signaling. Suppression of CXCR4 activity either by shRNA or pharmacological inhibition decreases MPNST cell growth in culture and inhibits tumorigenesis in allografts and in spontaneous genetic mouse models of MPNST. We further demonstrate conservation of these activated molecular pathways in human MPNSTs. Our findings indicate a role for CXCR4 in NF1-associated MPNST development and identify a therapeutic target.

Inducible Site-Specific Recombination in Neural Stem/Progenitor Cells

To establish a genetic tool for manipulating the neural stem/progenitor cell (NSC) lineage in a temporally controlled manner, we generated a transgenic mouse line carrying an NSC‐specific nestin promoter/enhancer expressing a fusion protein encoding Cre recombinase coupled to modified estrogen receptor ligand‐binding domain (ERT2). In the background of the Cre reporter mouse strain Rosa26lacZ, we show that the fusion CreERT2 recombinase is normally silent but can be activated by the estrogen analog tamoxifen both in utero, in infancy, and in adulthood. As assayed by β‐galactosidase activity in embryonic stages, tamoxifen activates Cre recombinase exclusively in neurogenic cells and their progeny. This property persists in adult mice, but Cre activity can also be detected in granule neurons and Bergmann glia at the anterior of the cerebellum, in piriform cortex, optic nerve, and some peripheral ganglia. No obvious Cre activity was observed outside of the nervous system. Thus, the nestin regulated inducible Cre mouse line provides a powerful tool for studying the physiology and lineage of NSCs. genesis 47:122–131, 2009.

Conditional ablation of brain-derived neurotrophic factor-TrkB signaling impairs striatal neuron development

Neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), are associated with the physiology of the striatum and the loss of its normal functioning under pathological conditions. The role of BDNF and its downstream signaling in regulating the development of the striatum has not been fully investigated, however. Here we report that ablation of Bdnf in both the cortex and substantia nigra depletes BDNF in the striatum, and leads to impaired striatal development, severe motor deficits, and postnatal lethality. Furthermore, striatal-specific ablation of TrkB, the gene encoding the high-affinity receptor for BDNF, is sufficient to elicit an array of striatal developmental abnormalities, including decreased anatomical volume, smaller neuronal nucleus size, loss of dendritic spines, reduced enkephalin expression, diminished nigral dopaminergic projections, and severe deficits in striatal dopamine signaling through DARPP32. In addition, TrkB ablation in striatal neurons elicits a non–cell-autonomous reduction of tyrosine hydroxylase protein level in the axonal projections of substantia nigral dopaminergic neurons. Thus, our results establish an essential function for TrkB in regulating the development of striatal neurons.

Neurofibromin modulates adult hippocampal neurogenesis and behavioral effects of antidepressants

Neurogenesis persists in the rodent dentate gyrus (DG) throughout adulthood but declines with age and stress. Neural progenitor cells (NPCs) residing in the subgranular zone of the DG are regulated by an array of growth factors and respond to the microenvironment, adjusting their proliferation level to determine the rate of neurogenesis. Here we report that genetic deletion of neurofibromin (Nf1), a tumor suppressor with RAS-GAP activity, in adult NPCs enhanced DG proliferation and increased generation of new neurons in mice. Nf1 loss-associated neurogenesis had the functional effect of enhancing behavioral responses to subchronic antidepressants and, over time, led to spontaneous antidepressive-like behaviors. Thus, our findings establish an important role for the Nf1-Ras pathway in regulating adult hippocampal neurogenesis, and demonstrate that activation of adult NPCs is sufficient to modulate depression- and anxiety-like behaviors.

Abstract A63: Identification of small molecules for GBM study

Glioblastoma Multiforme (GBM) is the most frequent of malignant tumors of the brain. It is fatal usually within fifteen months after diagnosis. The Cancer Genome Atlas (TCGA) has surveyed the genomes of hundreds of human GBMs for presence of mutations in genes previously associated with cancer. Among those identified, three of the five most frequently mutated genes encode the well-known tumor suppressors: P53, NF1, and Pten (TCGA, 2008). We have generated genetic mouse models of GBM based on mutation in these same three tumor suppressors and they develop GBM with 100% incidence. These mouse GBMs resemble the human counterparts by all commonly used criteria for diagnosis. Extensive study of these mice and tumors has provided novel insights into the natural history of GBM development and moreover pointed at the adult neural stem cell/progenitor compartment as the most likely and frequent source of these tumors (Kwon CH, et al., 2008). We have thus isolated a cancer stem cell and demonstrated a hierarchical growth for these tumors in vivo. (Chen J, et al., 2012) In an effort to identify novel GBM specific molecular pathways that might serve as therapeutic targets, we undertook a 200,000 small compound high throughput screen using primary low passage GBM cells generated by pooling cells from multiple mouse tumors (Mut6 cells). A critical counter screen was designed to minimize compounds that targeted the cell division and DNA replication machinery of cells since many normal cells divide and this property is not unique to cancer cells. We therefore ruled out all compounds that displayed toxicity to primary mouse embryo fibroblasts (MEFs) or to primary astrocytes. At the end of the screen, we selected three lead compounds with amenable physical-chemical properties for further evaluation and development as research discovery reagents and possible translational tools for drug development. We verified the activity of one of these compounds, termed Compound X, on mouse and human GBM primary cells as well as on a subset of established cancer cell lines, but not on MEFs or primary astrocytes. We identified a rapid cellular stress response accompanied by the robust activation of a transcription factor and eventual apoptotic response that can be mimicked by glucose deprivation. Through intensive structure-activity relationship study, analogs were successfully developed for in vivo activity and also for biochemical pull-down assay. Another compound (Compound Y) also induced apoptosis, however displaying response through completely different pathway, which indicates our screen identified multiple vulnerabilities in GBM tumor cells. Citation Format: Sang Kyun Lim, Yufeng Shi, Qiren Liang, Inga Nazarenko, Shuguang Wei, Maya Palnitkar, Yanjiao Li, Woosung Cho, Noelle Williams, Bruce Posner, Jef De Brabander, Luis F. Parada. Identification of small molecules for GBM study. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A63.

Abstract B63: Identification of small molecules that selectively target glioma stemlike cells

Gliomas are the most frequently occurring primary malignancies in the central nervous system, with glioblastoma multiforme (GBM) the most common and most aggressive of these tumors. GBM is virtually incurable because, following the standard treatment of care—surgical resection, radiation, and chemotherapy—the tumors eventually recur. Recent studies including our own indicate a crucial role for a specific subset of tumor cells (cancer stem cells) in tumor development and recurrence. We have generated fully penetrant, tumor-suppressor-based somatic mouse models of glioma, with gene mutations frequently found in human glioma (p53, NF1, Pten; termed Mut6 tumors). These tumor models develop GBM that histopathologically and molecularly mimics the human disease. Transcriptional profiling of these tumors revealed that they are most similar to human GBM. Using the pooled Mut6 stem-like cancer cells we performed a high-throughput screen designed to identify small molecules that inhibit the growth of these cells, as measured by a luminescence ATP assay. We performed a 200,000 compound library screen, initially identifying 4,480 compounds that were then retested in increasingly stringent assays such that we now have approximately 70 unique compounds that adversely affect glioma stem cell growth. These compound hits were found to have EC50s in the nanomolar range and to act with considerable specificity, affecting the mutant stem cells at concentrations that do not affect cultured astrocytes or mouse embryonic fibroblasts. Among these, we tested 46 compounds in metabolic stability tests and found 17 compounds with excellent stablilty profiles. We believe these compounds present promising lead compounds for drug development as well as for uncovering critical pathways in glioma tumor development and self renewal. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B63.