Kathleen Cato

Evidence for label-retaining tumour-initiating cells in human glioblastoma

Individual tumour cells display diverse functional behaviours in terms of proliferation rate, cell-cell interactions, metastatic potential and sensitivity to therapy. Moreover, sequencing studies have demonstrated surprising levels of genetic diversity between individual patient tumours of the same type. Tumour heterogeneity presents a significant therapeutic challenge as diverse cell types within a tumour can respond differently to therapies, and inter-patient heterogeneity may prevent the development of general treatments for cancer. One strategy that may help overcome tumour heterogeneity is the identification of tumour sub-populations that drive specific disease pathologies for the development of therapies targeting these clinically relevant sub-populations. Here, we have identified a dye-retaining brain tumour population that displays all the hallmarks of a tumour-initiating sub-population. Using a limiting dilution transplantation assay in immunocompromised mice, label-retaining brain tumour cells display elevated tumour-initiation properties relative to the bulk population. Importantly, tumours generated from these label-retaining cells exhibit all the pathological features of the primary disease. Together, these findings confirm dye-retaining brain tumour cells exhibit tumour-initiation ability and are therefore viable targets for the development of therapeutics targeting this sub-population.

NFIX Regulates Neural Progenitor Cell Differentiation During Hippocampal Morphogenesis

Neural progenitor cells have the ability to give rise to neurons and glia in the embryonic, postnatal and adult brain. During development, the program regulating whether these cells divide and self-renew or exit the cell cycle and differentiate is tightly controlled, and imbalances to the normal trajectory of this process can lead to severe functional consequences. However, our understanding of the molecular regulation of these fundamental events remains limited. Moreover, processes underpinning development of the postnatal neurogenic niches within the cortex remain poorly defined. Here, we demonstrate that Nuclear factor one X (NFIX) is expressed by neural progenitor cells within the embryonic hippocampus, and that progenitor cell differentiation is delayed within Nfix(-/-) mice. Moreover, we reveal that the morphology of the dentate gyrus in postnatal Nfix(-/-) mice is abnormal, with fewer subgranular zone neural progenitor cells being generated in the absence of this transcription factor. Mechanistically, we demonstrate that the progenitor cell maintenance factor Sry-related HMG box 9 (SOX9) is upregulated in the hippocampus of Nfix(-/-) mice and demonstrate that NFIX can repress Sox9 promoter-driven transcription. Collectively, our findings demonstrate that NFIX plays a central role in hippocampal morphogenesis, regulating the formation of neuronal and glial populations within this structure.

NFIX Regulates Proliferation and Migration Within the Murine SVZ Neurogenic Niche

Transcription factors of the nuclear factor one (NFI) family play a pivotal role in the development of the nervous system. One member, NFIX, regulates the development of the neocortex, hippocampus, and cerebellum. Postnatal Nfix(-/-) mice also display abnormalities within the subventricular zone (SVZ) lining the lateral ventricles, a region of the brain comprising a neurogenic niche that provides ongoing neurogenesis throughout life. Specifically, Nfix(-/-) mice exhibit more PAX6-expressing progenitor cells within the SVZ. However, the mechanism underlying the development of this phenotype remains undefined. Here, we reveal that NFIX contributes to multiple facets of SVZ development. Postnatal Nfix(-/-) mice exhibit increased levels of proliferation within the SVZ, both in vivo and in vitro as assessed by a neurosphere assay. Furthermore, we show that the migration of SVZ-derived neuroblasts to the olfactory bulb is impaired, and that the olfactory bulbs of postnatal Nfix(-/-) mice are smaller. We also demonstrate that gliogenesis within the rostral migratory stream is delayed in the absence of Nfix, and reveal that Gdnf (glial-derived neurotrophic factor), a known attractant for SVZ-derived neuroblasts, is a target for transcriptional activation by NFIX. Collectively, these findings suggest that NFIX regulates both proliferation and migration during the development of the SVZ neurogenic niche.

Heterozygosity for Nuclear Factor One X Affects Hippocampal-Dependent Behaviour in Mice

Identification of the genes that regulate the development and subsequent functioning of the hippocampus is pivotal to understanding the role of this cortical structure in learning and memory. One group of genes that has been shown to be critical for the early development of the hippocampus is the Nuclear factor one (Nfi) family, which encodes four site-specific transcription factors, NFIA, NFIB, NFIC and NFIX. In mice lacking Nfia, Nfib or Nfix, aspects of early hippocampal development, including neurogenesis within the dentate gyrus, are delayed. However, due to the perinatal lethality of these mice, it is not clear whether this hippocampal phenotype persists to adulthood and affects hippocampal-dependent behaviour. To address this we examined the hippocampal phenotype of mice heterozygous for Nfix (Nfix +/−), which survive to adulthood. We found that Nfix +/− mice had reduced expression of NFIX throughout the brain, including the hippocampus, and that early hippocampal development in these mice was disrupted, producing a phenotype intermediate to that of wild-type mice and Nfix−/− mice. The abnormal hippocampal morphology of Nfix +/− mice persisted to adulthood, and these mice displayed a specific performance deficit in the Morris water maze learning and memory task. These findings demonstrate that the level of Nfix expression during development and within the adult is essential for the function of the hippocampus during learning and memory.

In vivo overexpression of Emi1 promotes chromosome instability and tumorigenesis

Cell cycle genes are often aberrantly expressed in cancer, but how their misexpression drives tumorigenesis mostly remains unclear. From S phase to early mitosis, EMI1 (also known as FBXO5) inhibits the anaphase-promoting complex/cyclosome, which controls cell cycle progression through the sequential degradation of various substrates. By analyzing 7403 human tumor samples, we find that EMI1 overexpression is widespread in solid tumors but not in blood cancers. In solid cancers, EMI1 overexpression is a strong prognostic marker for poor patient outcome. To investigate causality, we generated a transgenic mouse model in which we overexpressed Emi1. Emi1-overexpressing animals develop a wide variety of solid tumors, in particular adenomas and carcinomas with inflammation and lymphocyte infiltration, but not blood cancers. These tumors are significantly larger and more penetrant, abundant, proliferative and metastatic than control tumors. In addition, they are highly aneuploid with tumor cells frequently being in early mitosis and showing mitotic abnormalities, including lagging and incorrectly segregating chromosomes. We further demonstrate in vitro that even though EMI1 overexpression may cause mitotic arrest and cell death, it also promotes chromosome instability (CIN) following delayed chromosome alignment and anaphase onset. In human solid tumors, EMI1 is co-expressed with many markers for CIN and EMI1 overexpression is a stronger marker for CIN than most well-established ones. The fact that Emi1 overexpression promotes CIN and the formation of solid cancers in vivo indicates that Emi1 overexpression actively drives solid tumorigenesis. These novel mechanistic insights have important clinical implications.

Expression of nuclear factor one A and -B in the olfactory bulb

The nuclear factor one (NFI) family of transcription factors consists of four members in vertebrates, NFIA, NFIB, NFIC, and NFIX, which share a highly conserved N‐terminal DNA‐binding domain. NFI genes are widely expressed in the developing mouse brain, and mouse mutants lacking NFIA, NFIB, or NFIX exhibit developmental deficits in several areas, including the cortex, hippocampus, pons, and cerebellum. Here we analyzed the expression of NFIA and NFIB in the developing and adult olfactory bulb (OB), rostral migratory stream (RMS), and subventricular zone (SVZ). We found that NFIA and NFIB are expressed within these regions during embryonic and postnatal development and in the adult. Immunohistochemical analysis using cell‐type‐specific markers revealed that migrating neuroblasts in the adult brain express NFI transcription factors, as do astrocytes within the RMS and progenitor cells within the SVZ. Moreover, astrocytes within the OB express NFIA, whereas mitral cells within the OB express NFIB. Taken together these data show that NFIA and NFIB are expressed in both the developing and the adult OB and in the RMS and SVZ, indicative of a regulatory role for these transcription factors in the development of this facet of the olfactory system. J. Comp. Neurol., 520:3135–3149, 2012.

Abstract 3586: Overexpression of Emi1 causes chromosomal instability and cancer

Chromosomal Instability (CIN) is a common occurrence in solid cancer associated with poor survival in patients. A significant percentage of these tumors show abnormal chromosomes with near-triploid or tetraploid numbers exhibiting numerical CIN (nCIN). nCIN is caused mainly due to deregulation of cell cycle proteins. Early Mitotic Inhibitor 1 (Emi1) is a cell cycle protein that regulates the activity of E3 ubiquitin ligase, Anaphase Promoting Complex/Cyclosome (APC/C), which targets many cell cycle proteins for degradation. Emi1 is overexpressed in many solid tumors but not blood cancers. Unpublished data from our lab has shown that Emi1 overexpression in mouse models causes more penetrant and metastatic tumor than control tumors. Breast cancer tissues show significantly high expression of Emi1 at protein level as compared to control breast tissue samples, in agreement with the microarray data from The Cancer Genome Atlas (TCGA). In addition, Emi1 expression from breast cancer TCGA data strongly correlated with the chromosomal instability signature than many of the well-established genes known to promote CIN phenotype. To understand the mechanisms of CIN due to Emi1 overexpression, we overexpressed Emi1 in HeLa cells expressing GFP-tagged histone protein to monitor the cells during mitosis by live cell imaging. HeLa cells overexpressing Emi1 show CIN following delays in both chromosome alignment at the metaphase plate and, subsequently, anaphase onset (p = 0.0008 and p = 0.0028). There is a significant increase in CIN phenotypes and abnormal mitoses such as anaphase bridges/lagging chromosomes, mitotic arrest and cell death. These results indicate that Emi1 overexpression actively drives tumorigenesis. Citation Format: Srividya Vaidyanathan, Kathleen Cato, Sandra Pavey, Nikolas K. Haass, Brian G. Gabrielli, Pascal HG Duijf. Overexpression of Emi1 causes chromosomal instability and cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3586.