Hui Zong

Mosaic Analysis with Double Markers Reveals Tumor Cell of Origin in Glioma

Cancer cell of origin is difficult to identify by analyzing cells within terminal stage tumors, whose identity could be concealed by the acquired plasticity. Thus, an ideal approach to identify the cell of origin is to analyze proliferative abnormalities in distinct lineages prior to malignancy. Here, we use mosaic analysis with double markers (MADM) in mice to model gliomagenesis by initiating concurrent p53/Nf1 mutations sporadically in neural stem cells (NSCs). Surprisingly, MADM-based lineage tracing revealed significant aberrant growth prior to malignancy only in oligodendrocyte precursor cells (OPCs), but not in any other NSC-derived lineages or NSCs themselves. Upon tumor formation, phenotypic and transcriptome analyses of tumor cells revealed salient OPC features. Finally, introducing the same p53/Nf1 mutations directly into OPCs consistently led to gliomagenesis. Our findings suggest OPCs as the cell of origin in this model, even when initial mutations occur in NSCs, and highlight the importance of analyzing premalignant stages to identify the cancer cell of origin.

Mosaic Analysis with Double Markers in Mice

We describe a method termed MADM (mosaic analysis with double markers) in mice that allows simultaneous labeling and gene knockout in clones of somatic cells or isolated single cells in vivo. Two reciprocally chimeric genes, each containing the N terminus of one marker and the C terminus of the other marker interrupted by a loxP-containing intron, are knocked in at identical locations on homologous chromosomes. Functional expression of markers requires Cre-mediated interchromosomal recombination. MADM reveals that interchromosomal recombination can be induced efficiently in vivo in both mitotic and postmitotic cells in all tissues examined. It can be used to create conditional knockouts in small populations of labeled cells, to determine cell lineage, and to trace neuronal connections. To illustrate the utility of MADM, we show that cerebellar granule cell progenitors are fated at an early stage to produce granule cells with axonal projections limited to specific sublayers of the cerebellar cortex.

Pro-neural miR-128 is a glioma tumor suppressor that targets mitogenic kinases

MicroRNAs (miRNAs) carry out post-transcriptional control of a multitude of cellular processes. Aberrant expression of miRNA can lead to diseases, including cancer. Gliomas are aggressive brain tumors that are thought to arise from transformed glioma-initiating neural stem cells (giNSCs). With the use of giNSCs and human glioblastoma cells, we investigated the function of miRNAs in gliomas. We identified pro-neuronal miR-128 as a candidate glioma tumor suppressor miRNA. Decreased expression of miR-128 correlates with aggressive human glioma subtypes. With a combination of molecular, cellular and in vivo approaches, we characterize miR-128s tumor suppressive role. miR-128 represses giNSC growth by enhancing neuronal differentiation. miR-128 represses growth and mediates differentiation by targeting oncogenic receptor tyrosine kinases (RTKs) epithelial growth factor receptor and platelet-derived growth factor receptor-α. Using an autochthonous glioma mouse model, we demonstrated that miR-128 repressed gliomagenesis. We identified miR-128 as a glioma tumor suppressor that targets RTK signaling to repress giNSC self-renewal and enhance differentiation.

Site-specific integrase-mediated transgenesis in mice via pronuclear injection

Microinjection of recombinant DNA into zygotic pronuclei has been widely used for producing transgenic mice. However, with this method, the insertion site, integrity, and copy number of the transgene cannot be controlled. Here, we present an integrase-based approach to produce transgenic mice via pronuclear injection, whereby an intact single-copy transgene can be inserted into predetermined chromosomal loci with high efficiency (up to 40%), and faithfully transmitted through generations. We show that neighboring transgenic elements and bacterial DNA within the transgene cause profound silencing and expression variability of the transgenic marker. Removal of these undesirable elements leads to global high-level marker expression from transgenes driven by a ubiquitous promoter. We also obtained faithful marker expression from a tissue-specific promoter. The technique presented here will greatly facilitate murine transgenesis and precise structure/function dissection of mammalian gene function and regulation in vivo.

Genetic Mosaic Dissection of Lis1 and Ndel1 in Neuronal Migration

Coordinated migration of newly born neurons to their prospective target laminae is a prerequisite for neural circuit assembly in the developing brain. The evolutionarily conserved LIS1/NDEL1 complex is essential for neuronal migration in the mammalian cerebral cortex. The cytoplasmic nature of LIS1 and NDEL1 proteins suggest that they regulate neuronal migration cell autonomously. Here, we extend mosaic analysis with double markers (MADM) to mouse chromosome 11 where Lis1, Ndel1, and 14-3-3ɛ (encoding a LIS1/NDEL1 signaling partner) are located. Analyses of sparse and uniquely labeled mutant cells in mosaic animals reveal distinct cell-autonomous functions for these three genes. Lis1 regulates neuronal migration efficiency in a dose-dependent manner, while Ndel1 is essential for a specific, previously uncharacterized, late step of neuronal migration: entry into the target lamina. Comparisons with previous genetic perturbations of Lis1 and Ndel1 also suggest a surprising degree of cell-nonautonomous function for these proteins in regulating neuronal migration.

The cellular origin for malignant glioma and prospects for clinical advancements

Glioma remains incurable despite great advancements in medicine. Targeting the cell of origin for gliomas could bring great hope for patients. However, as a collection of diverse diseases, each subtype of glioma could derive from a distinct cell of origin. To resolve such a complex problem, one must use multiple research approaches to gain deep insights. Here we review current evidence regarding the cell of origin from clinical observations, whole-genome molecular pathology and glioma animal models. We conclude that neural stem cells, glial progenitors (including oligodendrocyte progenitor cells) and astrocytes could all serve as cells of origin for gliomas, and that cells incurring initial mutations (cells of mutation) might not transform, while their progeny cells could instead transform and act as cells of origin. Further studies with multidisciplinary approaches are needed to link each subtype to a particular cell of origin, and to develop effective therapies that target the signaling network within these cells.

Modeling sporadic loss of heterozygosity in mice by using mosaic analysis with double markers (MADM)

The initiation and progression of many human cancers involve either somatic activation of protooncogenes or inactivation of tumor-suppressor genes (TSGs) in sporadic cells. Although sporadic gain-of-function of protooncogenes has been successfully modeled in mice [e.g., Johnson L, Mercer K, Greenbaum D, Bronson RT, Crowley D, Tuveson DA, Jacks T (2001) Nature 410:1111–1116], generating a similar degree of sparseness of TSG loss-of-function remains a challenge. Here, we use mosaic analysis with double markers (MADM) to achieve TSG inactivation and concurrent labeling in sporadic somatic cells of mice, closely mimicking loss of heterozygosity as occurs in human cancers. As proof of principle, we studied the consequence of sporadic loss of p27kip1, a cyclin-dependent kinase inhibitor. MADM-mediated loss of p27kip1 results in mutant cell expansion markedly greater than that observed in conventional p27kip1 knockouts. Moreover, the direct comparison of WT and mutant cells at single-cell resolution afforded by MADM reveals that p27kip1 regulates organ size in vivo by cell-autonomous control of cell cycle exit timing. These studies establish MADM as a high-resolution method for modeling sporadic loss of heterozygosity in mice, providing insights into TSG function.

Cell of Origin for Malignant Gliomas and Its Implication in Therapeutic Development

Malignant glioma remains incurable despite tremendous advancement in basic research and clinical practice. The identification of the cell(s) of origin should provide deep insights into leverage points for one to halt disease progression. Here we summarize recent studies that support the notion that neural stem cell (NSC), astrocyte, and oligodendrocyte precursor cell (OPC) can all serve as the cell of origin. We also lay out important considerations on technical rigor for further exploring this subject. Finally, we share perspectives on how one could apply the knowledge of cell of origin to develop effective treatment methods. Although it will be a difficult battle, victory should be within reach as along as we continue to assimilate new information and facilitate the collaboration among basic scientists, translational researchers, and clinicians.

Transformation of quiescent adult oligodendrocyte precursor cells into malignant glioma through a multistep reactivation process

Significance How malignant gliomas arise in a mature brain remains a mystery, which hinders the development of effective treatments. Which cell types can escape their quiescent, adult state and how they do so is unknown. Additionally, because gliomas are only detected at advanced stages, the full course of transformation remains uncharacterized. Here we report that adult oligodendrocyte precursor cells, despite their relatively quiescent properties, can be reactivated to a highly proliferative state by p53 and NF1 mutations and give rise to malignant gliomas. Furthermore, we describe the early phase of gliomagenesis for the first time, revealing a multistep process of reactivation, dormancy, and final transformation in which mammalian target of rapamycin signaling plays a critical role at both early and late steps. How malignant gliomas arise in a mature brain remains a mystery, hindering the development of preventive and therapeutic interventions. We previously showed that oligodendrocyte precursor cells (OPCs) can be transformed into glioma when mutations are introduced perinatally. However, adult OPCs rarely proliferate compared with their perinatal counterparts. Whether these relatively quiescent cells have the potential to transform is unknown, which is a critical question considering the late onset of human glioma. Additionally, the premalignant events taking place between initial mutation and a fully developed tumor mass are particularly poorly understood in glioma. Here we used a temporally controllable Cre transgene to delete p53 and NF1 specifically in adult OPCs and demonstrated that these cells consistently give rise to malignant gliomas. To investigate the transforming process of quiescent adult OPCs, we then tracked these cells throughout the premalignant phase, which revealed a dynamic multistep transformation, starting with rapid but transient hyperproliferative reactivation, followed by a long period of dormancy, and then final malignant transformation. Using pharmacological approaches, we discovered that mammalian target of rapamycin signaling is critical for both the initial OPC reactivation step and late-stage tumor cell proliferation and thus might be a potential target for both glioma prevention and treatment. In summary, our results firmly establish the transforming potential of adult OPCs and reveal an actionable multiphasic reactivation process that turns slowly dividing OPCs into malignant gliomas.

Loop 6 of RhoA confers specificity for effector binding, stress fiber formation, and cellular transformation.

Rho family GTPases regulate multiple cellular processes, including cytoskeletal organization, gene expression, and transformation. These effects are achieved through the interaction of GTP-bound proteins with various downstream targets. A series of RhoA/Rac1 and Rho/Ras chimeras was generated to map the domain(s) of RhoA involved in its association with two classes of effector kinase, represented by PRK2 and ROCK-I. Although the switch 1 domain was required for effector binding, the N terminus of Rho (residues 1–75) was interchangeable with that of Rac. This suggested that the region of Rho that confers effector binding specificity lay further C-terminal. Subsequent studies indicated that the “insert domain”(residues 123–137), a region unique to Rho family GTPases, is not the specificity determinant. However, a determinant for effector binding was identified between Rho residues 75–92. Rac to Rho point mutations (V85D or A88D) within loop 6 of Rac promoted its association with PRK2 and ROCK, whereas the reciprocal Rho(D87V/D90A) double mutant significantly reduced effector binding capacity. In vivostudies showed that microinjection of Rac(Q6IL/V85D/A88D) but not Rac(Q6IL) induced stress fiber formation in LLC-PK epithelial cells, suggesting that loop 6 residues conferred the ability of Rac to activate ROCK. On the other hand, the reciprocal Rho (Q6IL/D87V/D90A) mutant was defective in its ability to transform NIH 3T3 cells. These data suggest that although Rho effectors can utilize a Rho or Rac switch 1 domain to sense the GTP-bound state of Rho, unique residues within loop 6 are essential for determining both effector binding specificity and cellular function.