Molecular subclassification, stem cell markers and growth regulatory pathways in gliomas

Abstract

Glioblastoma is the most common and aggressive primary brain tumor in adults. Median survival is no more than 15 months, even with combined treatment regimens. The lack of accurate pre-clinical model systems has limited the development of new treatment options for this deadly disease.

In the first study we set out with the goal of creating improved research models for studying the disease. To this end we generated a glioblastoma patient derived cell line (PDCL) library. PDCLs grown under neural stem cell conditions functionally isolate glioblastoma cells with stem-like properties. In keeping with their cancer stem cell like nature PDCLs readily formed patient derived xenografts (PDXs) when transplanted in vivo, recapitulating histological features and maintaining genetic/protein profiles of founding tumors. Expression profiling of PDCLs revealed 4 in vitro subclasses differentially associated with WNT/B-catenin, TGF-B, BMP, Interferon, Notch and p53 signaling. Applying these new in vitro PDCL-classes to glioblastoma patient data generated clinically relevant subclasses with differences in age and survival. Hence, new PDCL models represent the original disease including aspects such as genomics/proteomics, intratumoral heterogeneity and cancer stem cell biology. In addition to functional isolation of stem-like cells using PDCL methods there have been several proteins proposed to distinguish cancer stem cells (CSCs). The most popular such protein is Prominin- 1 (PROM1/CD133). In the second study we investigated Prominin-1 expression in the developing CNS and glioblastoma. Prominin-1 is expressed in the ventricular zone of the embryonic brain and postnatally shifts to a pattern of distributed cells. The adult murine Prom1+ cell population mainly consists of Olig2+ slow cycling glial cells. Further, we established that Prom1 is independent of Olig2. Human normal brain PROM1 is associated with GFAP and SOX2. In glioblastoma PDCLs PROM1+ cells can express GFAP, SOX2 and OLIG2. PROM1 patterns are very heterogeneous across PDCLs and patient samples revealed that high PROM1 expression is less common in IDH1 mutant samples. In the third and fourth studies we investigated the possible functions of NPM1 in glioblastoma. NPM1 is a nucleolar chaperone protein highly upregulated in glioblastoma tissue samples and cell lines. We showed that NPM1 regulated the morphology of the nucleolus. This is likely due to NPM1 interacting with proteins implicated in the organization of chromatin such as HP1γ, H1.5 and H3. We also found that NPM1/DNMT3A co-depletion induced rDNA transcription and nucleolar normalization. Although DNMT3A loss upregulated rDNA transcription on its own depletion of NPM1 increased this effect suggesting NPM1 loss creates a permissive environment for further epigenetic change. NPM1 depletion sensitized glioblastoma cell lines to chemotherapy and further insults to chromatin stability such as the co-depletion of H1.5, implying NPM1 as a novel drug target.

This thesis characterizes a glioblastoma PDCL library, illustrating the benefits of these new research models and addresses the complex expression of Prominin-1 and function of NPM1 with regards to molecular subclasses and growth regulatory pathways in glioblastoma, respectively.