Malignant glioma is one of the deadliest types of cancer. Understanding how the cell of origin progressively evolves toward malignancy in greater detail could provide mechanistic insights and lead to novel concepts for tumor prevention and therapy. Previously we have identified oligodendrocyte precursor cell (OPC) as the cell of origin for glioma following the concurrent deletion of p53 and NF1 using a mouse genetic mosaic system that can reveal mutant cells prior to malignancy. In the current study, we set out to deconstruct the gliomagenic process in two aspects. First, we determined how the individual loss of p53 or NF1 contributes to aberrant behaviors of OPCs. Second, we determined how signaling aberrations in OPCs progressively change from pre-malignant to transformed stages. We found that while the deletion of NF1 leads to mutant OPC expansion through increased proliferation and decreased differentiation, the deletion of p53 impairs OPC senescence. Signaling analysis showed that, while PI3K and MEK pathways go through stepwise over-activation, mTOR signaling remains at the basal level in pre-transforming mutant OPCs but is abruptly up-regulated in tumor OPCs. Finally, inhibiting mTOR via pharmacological or genetic methods, led to a significant blockade of gliomagenesis but had little impact on pre-transforming mutant OPCs, suggesting that mTOR is necessary for final transformation but not early progression. In summary, our findings show that deconstructing the tumorigenic process reveals specific aberrations caused by individual gene mutations and altered signaling events at precise timing during tumor progression, which may shed light on tumor-prevention strategies.
Glioma is currently an incurable disease. Identifying the cell of origin for glioma could provide critical insights for developing effective therapies. We previously used a mouse genetic system termed Mosaic Analysis using Double Markers (MADM) to introduce p53 and NF1 mutations into neural stem cells to determine which cell type is responsible for gliomagenesis. The unequivocal GFP-labeling of sparse mutant cells generated by MADM enabled us to study the entire course of tumor development, from pre-transforming stages to full malignancy. Based on thorough histological, transcriptomic, and genetic analyses we identified oligodendrocyte precursor cells (OPCs) as the cell of origin in this glioma model. Here, we sought to understand the distinct role of p53 and NF1 in the transformation of OPCs since understanding these roles can give us a more thorough insight into how future therapies may be designed. The MADM system generates sparse GFP+ null and RFP+ sibling WT OPCs in an otherwise colorless, heterozygous mouse, allowing us to assess the impact of TSG loss on the capacity of cells to proliferate, survive, and differentiate. The loss of NF1 alone significantly increased mutant OPC proliferation with a proportional decrease in differentiation. However, massively over-expanded NF1-null OPCs did not transform into malignant gliomas, implicating the critical role of p53 in tumor suppression. Surprisingly, the loss of p53 alone had no effect on mutant OPC number, proliferation, or differentiation, suggesting that p53 function as a braking system that won't manifest its activity in the absence of driver mutation, such as NF1 loss. Next, we set out to test whether restoring either TSG function in tumor OPC cell lines would have any therapeutic efficacy. The restoration of wild type p53 led to a significant increase in both cell-cycle arrest and cell death. Since p53 function is commonly altered from mutations in patients, rather than complete loss of the protein, we tested whether restoring mutant p53 function with PRIMA-1 would have the same biological response as WT p53. We found that the restoration of mutant p53 function resulted in similar levels of cell cycle arrest and cell death as WT p53. This finding suggests that rescuing p53 function using small molecules may be of high value for future therapies. It also suggests that the loss of p53 activity is not a permanent fixture of tumor cells but that p53 can still exert it's tumor suppressor activity after transformation. Next we tested the therapeutic effects of the restoration of the GAP domain of NF1, which should reduce Ras activity in tumor OPCs. We found that the expression of WT but not the “GAP-dead” NF1-GAP led to a decrease in proliferation and an increase in differentiation of glioma cells. To test pharmacological compounds, we explore the possibility of targeting downstream effectors of Ras signaling, specifically mTOR inhibition because phospho-Akt level is elevated in tumor OPCs compared to WT OPCs. When we inhibited mTOR activity in MADM tumor mice at an age that is critical for mutant OPC expansion prior to transformation, we found that it blocked the ability of mutant OPCs to over-expand, suggesting that mTOR inhibition could potentially prevent gliomagenesis. In summary our data demonstrates that while NF1 loss increases OPC proliferation and decreases differentiation, p53 loss is critical for transformation and that restoring the function of either of these pathways has profound effects on tumor OPC biology that could be translated into future therapeutic strategies. Citation Format: Phillippe P. Gonzalez, Hui Zong. Deciphering individual roles of p53 and NF1 in the cell of origin for malignant glioma and the implications of targeted therapy. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B16.
Cancer is difficult to treat because it is a disease caused by multiple genetic alterations. Understanding how individual mutations contribute to pathogenesis and how they cooperate to aid malignancy offers deep insights of tumorigenic mechanisms that will facilitate the design of effective treatment. However, conventional methods, including most mouse cancer models, do not offer the temporal and spatial resolution required to study tumorigenesis in great details, especially because single gene mutations rarely lead to detectable malignancy. To resolve this issue, our lab uses a novel mouse model termed MADM (Mosaic Analysis with Double Markers) to analyze the contribution of individual mutations to aberrant behaviors of tumor cell of origin during pre-transforming stages. Starting with a mouse heterozygous for a TSG, MADM can generate sparse mutant cells that are null for the TSG via Cre-loxP mediated inter-chromosomal mitotic recombination. Concurrently, MADM unambiguously labels mutant cells with GFP and their wild-type sibling cells with RFP, allowing us to precisely analyze behavioral changes of green mutant cells, using their sibling red cells as the internal control. Using MADM to model glioblastoma, the most common and aggressive malignant primary brain tumor, our lab previously demonstrated that oligodendrocyte precursor cell (OPC) is the cell of origin. Using MADM to generate p53 and NF1 double-null OPCs, we found that mutant OPCs dramatically over-expanded at the pre-transforming stage compared to WT sibling cells. Further analysis indicated that mutant OPCs manifest augmented proliferative activities and halted differentiation processes. However, it is still unknown what is the individual contribution of the loss of p53 and NF1 and how their cooperativity leads to malignant transformation. Utilizing MADM, we generated genetic mosaic mice with individual loss of either p53 or NF1 in OPCs and determined the cellular property changes of mutant cells, including proliferative rate, differentiation ability, overall expansion, and malignant transformation. We found that, while the mosaic loss of NF1 alone is sufficient to lead to increased proliferation and impaired differentiation of mutant cells, it is insufficient to lead to malignancy even though mutant cells overwhelm the mouse brain, suggesting that p53 plays critical gatekeeping roles in gliomagenesis. On the other hand, when we examined brains containing p53-null OPCs, we were surprised to find that the loss of p53 alone had no detectable effect on cell number, proliferation and differentiation of mutant cells. We reasoned that the tumor suppressing functions of p53 may only be manifested in an NF1 mutant background, and thus generated a new mosaic model in which all OPCs are NF1-null while green cells are p53 null and red ones are p53 WT. Surprisingly, we still found no differences in cell number, proliferation, or differentiation between green and red cells, suggesting that p53 suppress gliomagenesis through some unknown mechanisms. In summary, our data suggested that NF1 loss leads to glioma initiation by promoting proliferation and inhibiting differentiation of OPCs while p53 loss only contributes to later stage of glioma progressions, and that an effective treatment paradigm would need to address all these mechanisms to halt malignancy. Citation Format: Phillippe Gonzalez, Chong Liu, Hui Zong. Deciphering individual roles of p53 and NF1 in suppressing gliomagenesis. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B14.
Glioblastoma remains one of the most deadly cancers due to their rapid onset and the limited effectiveness of therapies. Here we use a mouse genetic system termed Mosaic Analysis with Double Markers (MADM) to study progression towards malignancy in oligodendrocyte progenitor cells (OPC), the cell of origin of glioma. We use gene deletions to uncover individual roles of two commonly mutated tumor suppressor genes, p53 and NF1. NF1 acts as a negative regulator of OPC self-renewal and promoter of OPC differentiation, and p53 increases the permanent arrest of OPC proliferation during times of stress. Subsequent analysis revealed that the downstream NF1 effector, mTOR, is critical for OPC transformation. Furthermore, mutant OPCs expand at the expense of surrounding non-mutant OPCs through a mechanism termed cell competition, to maintain proper density in the brain. This cell competition phenomenon is critical for OPC transformation as the complete inhibition of this property leads to inhibition of gliomagenesis irrespective of p53 and NF1 mutations. In summary, our findings reveal distinct roles for p53 and NF1 during gliomagenesis and a unique cell-cell interaction during the progression that is critical for malignancy.
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