Funded Grants

Promoter-based isolation and assessment of differential gene expression by stage-defined glial tumor stem cells

Over the past decade, it has become clear that some tumors may arise from cancerous tissuespecific stem or progenitor cells, that may remain distinct and separable from their neoplastic derivatives. The cancer stem cell hypothesis, as formulated in systems as diverse as blood, brain and breast cancers, argues that cancers are sustained by expansion from relatively small numbers of neoplastic progenitors 29,30. In the brain, it has been clear for several years that many types of CNS tumors, including periventricular tumors, medulloblastomas in children, and gliomas in adults, exhibit multipotentiality and self-renewal in culture 31, suggesting either their derivation from, or de-differentiation to, a stem cell10. However, the stem cell hypothesis goes further in emphasizing the critical dependence of tumor expansion on persistent stem cells. Specifically, it argues that tumor daughter cells, which may comprise much or most of the tumor bulk, are not necessarily capable of self-renewing divisions and clonal expansion 32. As such, they may be incapable of passaging or propagating the tumor, in the absence of their parental tumor stem cells. Evidence for this position has been offered in glioma, for which a discrete pool identifiable by AC133- immunoreactivity was reported to be able to initiate and propagate tumor expansion in NOD-SCID immunodeficient recipients, while the AC133-depleted remainder was incapable of doing so23,32. It is the latter point that is remarkable here, in that it suggests that the majority of a tumor is comprised of daughter cells unable to independently propagate their parental tumor. On this basis, the central role of a neoplastic progenitor in initiating brain cancers, and in regenerating them after cytotoxic treatment, seems both of clear import and a formidable therapeutic challenge – it requires us to understand the molecular regulation of relatively uncommon tumor progenitors within a much larger tumor mass, as a necessary step in the definitive treatment of that tumor.

We propose here to approach this issue from the standpoint of understanding the differences between tumor stem and progenitor cells, and the native stem and progenitor cells from which they likely arise in vivo. Over the past two decades, we and many others have reported that the mature brain continues to harbor a population of neural stem cells, multipotential and self-renewing cells that persist from early development 6,16,33-37. In the adult forebrain, these cells remain in a discrete layer, the subependyma, which lines the lateral ventricles. The adult brain also harbors a number of different types of more restricted progenitor cells, that can give rise to new neurons and glial cells, the latter including the two major glial cell types, oligodendrocytes and astrocytes. Whereas mature human neurons and oligodendrocytes are incapable of further cell division, the stem and progenitor cells that give rise to them can divide throughout life. These precursor cells thus appear likely candidates as sources for primary tumors of the central nervous system10. This may be especially true in children, in whom periventricular tumors predominate. But even in adults, in whom glial tumors are much more common, tumors may arise from resident glial progenitor cells of the tissue parenchyma, rather than from mature glial cells. In this proposal, we seek to use a genetic strategy of promoter-based selection to isolate stem and progenitor cells from different stage-defined adult glial tumors, in order to study the biology and genomics of the neoplastic progenitors from which these tumors arise.

In studies of the past 7 years, we have used fluorescent reporter genes placed under the control of progenitor cell-specific promoters to identify and then isolate neural stem cells24, neuronal progenitors2,3, as well as parenchymal glial progenitor cells4,5, from both fetal and adult human brain tissues. Recently we have extended this analysis to CNS tumors, for the purpose of isolating stem and progenitor cells from tumors, as a first step towards understanding how these cells differ in their biology and gene expression from normal, non-neoplastic adult progenitor cells. Our pilot studies thus far have included neurocytoma, oligodendroglioma, mixed oligo-astrocytoma and anaplastic astrocytoma, and have proven both encouraging and fundamentally exciting: Using promoter-based selection, we have now been able to extract neoplastic progenitors from each of these tumors, that correspond to ontogenetically-analogous progenitors in the normal brain. This has allowed us to begin comparing the gene expression patterns of these tumor stem cells to their native, non-transformed analogues.

In our first formal validation of this strategy, we used Affymetrix arrays to compare the gene expression profile of neurocytoma, a predominantly pro-neuronal tumor of the ventricular wall, to that of the normal adult subependyma, as well as to that of neural progenitor cells sorted from normal brain on the basis of nestin enhancer-specified, GFP-based FACS. We found that this analysis revealed a discrete and relatively small set of highly differentially expressed genes in the neurocytoma cells relative to normal ventricular zone and its nestin-selected progenitors22. Among the differentially expressed genes, the growth hormone IGF2 and its downstream signal intermediates were prominent; indeed, their selective overexpression was quantitatively remarkable: qPCR confirmed a >50-fold increase in IGF signal components in neurocytoma cells compared to native subependymal progenitors, despite substantially overlapping expression patterns otherwise. Immunocytochemistry and Western analysis confirmed high IGF2 expression levels by neurocytoma, and pathway analysis using Ingenuity confirmed the differential transcriptional activation of the IGF2 signaling pathway. We are now in the midst of testing IGF2 antagonists to assess their therapeutic potential in these tumors. Yet whether IGF2 signal inhibition is found to be a potential treatment strategy in neurocytoma or not, the value of this strategy of comparing the expression profiles of tumor progenitors to their sorted native homologues, as a means of rapidly and efficiently identifying potentially causal genes in neural tumor formation, and in identifying the causal stem or progenitor cell in a given heterogeneous cancer, now seems clear.

In this proposal we seek to follow this approach in assessing adult glial tumors, with the goal of identifying those genes associated with the transformation of a native glial progenitor into a glioma. Using progenitor cell isolates specifically extracted from human astrocytomas, oligodendroglioma, and mixed astrocytoma-oligodendrogliomas, we will first identify which are actually tumorigenic, by transplanting each promoter-defined isolate into naïve immunodeficient mice to establish which progenitor isolates are able to initiate tumors, and what types of tumors are generated by each purified phenotype. We will then test the possibility that by subtracting the gene expression profile of normal, native progenitors from that of their tumor-derived counterparts, we can identify those genes that are specifically dysregulated in tumor progenitors. In particular, for each of the glial progenitor cell types to be investigated, we expect to identify those genes overexpressed by tumorigenic vs. normal progenitors, and then use this information to predict those signaling pathways differentially active in the tumor progenitors. We thereby hope to identify a discrete cohort of genes and pathways that distinguish each tumor progenitor cell type from its counterpart in normal brain.

The importance of defining those genes and signaling pathways that distinguish the transformed progenitors of brain tumors from the native progenitor cells from which they derive is manifold. The different phenotypes of human brain tumors may comprise a hierarchy of neoplastic progenitor cells, with distinct tumors corresponding to the transformed derivatives of cells transformed at distinct points in their lineage progression. As a result, the daughter cells of transformed progenitor cells may be sufficiently distinct from their parents in both their expansion and growth control, that therapy directed at their derivative tumors may be neither appropriate nor effective against the parental transformed progenitor clone. Of note, this differs from the tumor stem cell hypothesis, which would argue that abolition of the parental progenitor, or tumor stem cell, would be sufficient to destroy a given tumor, whose derivative phenotypes would be incapable of self-renewal. Rather, we suggest that derivative phenotypes may well be capable of autonomous self-renewal once generated, but that cytotoxic therapy directed against them, without abolition of their underlying parental transformant, is destined for relapse and ultimate treatment failure. The therapeutic implication is that oncolytic therapy may need to be directed not solely at either the tumor stem cell or its derivatives, but rather at both, through fundamentally different mechanistic strategies. By the strategies outlined in this proposal, we hope to efficiently develop anti-neoplastic strategies directed specifically at genes critical to the growth and survival of tumor stem cells. By separately targeting tumor stem cells and their daughter cells with mechanistically distinct agents, we hope to minimize the likelihood of clinical relapse after initial treatment of malignant glioma. By so doing, we hope to provide clinical benefit to a patient population whose prognosis remains as dire today as it was decades ago, long before the advent of the technologies and approaches that we now hope to bring to bear to the treatment of this disease.

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