A nucleolar protein, nucleostemin, is now implicated in the control of stem and cancer cell proliferation. Nucleostemin may modulate p53 function through shuttling between the nucleolus and the nucleoplasm.
Stem cells are present throughout embryonic development as well as in several organs of the adult organism. They constitute a pool of undifferentiated cells with the remarkable ability to perpetuate through self-renewal while also retaining the potential to terminally differentiate into various mature cell types. Coordinated control of self-renewal and commitment to differentiation is key to maintaining the homeostasis of the stem cell compartment, which when deregulated may contribute to cancer pathogenesis1. The identification of stem-cell−specific proteins and the elucidation of novel regulatory pathways that ensure the integration of these processes are therefore of fundamental importance.
In the December 1 issue of Genes & Development, Tsai and McKay describe the isolation and characterization of nucleostemin, a novel p53 binding protein localized in the nucleoli of stem cells and cancer cells, but absent from committed and terminally differentiated cells2. Although it has been known for decades that the nucleolus is the subnuclear compartment where rRNA transcription and ribosome assembly occur, the notion that this subnuclear structure functions as a mere 'ribosome factory' has recently been challenged. More recent findings unraveled a new function for the nucleolus as a storehouse for the titration of specific proteins and the consequent modulation of their molecular pathways3. Regulation of the tumor-suppressor p53 highlights this nucleolar task. Retention of a negative regulator of p53, the oncoprotein MDM2 (ref. 4), in the nucleolus results in stabilization of nucleoplasmic p53 (ref. 5). Nucleolar retention of MDM2 is promoted by the tumor-suppressor ARF, a nucleolar protein increasingly expressed as cells withdraw from the cell cycle when maintained in culture or upon oncogenic stress6. The work of McKay and Tsai, then, loosely places nucleostemin in a defined molecular network.
The authors identified nucleostemin as a cDNA expressed in rat stem cells in the central nervous system (CNS). The nucleostemin protein, although prominent in the nucleoli of undifferentiated cells (for example, embryonic stem cells) and various cancer cell lines, was also found in the nucleoplasm, albeit at lower levels. Nucleostemin was markedly downregulated during cellular differentiation both throughout embryonic development and in adulthood. In vivo, nucleostemin was expressed preferentially in neuroepithelial precursors during embryogenesis (Fig. 1) and shut off during CNS differentiation. The protein was also expressed in adult bone marrow hematopoietic stem cells, but not in committed B-lymphocytes and granulocytes. In culture, nucleostemin became undetectable upon induction of differentiation in CNS rat stem cells. Notably, during CNS development in vivo, nucleostemin downregulation preceded the downregulation of proteins that mark cell cycle exit. Similarly, nucleostemin was downregulated in CNS rat stem cells before cell cycle exit. These data indicate that nucleostemin downregulation may lead to cell cycle exit rather than occur as the consequence of cellular differentiation and cell cycle withdrawal. Indeed, nucleostemin knockdown experiments in CNS stem cells and in U2OS cancer cells resulted in an increase in non-cycling cells.
Figure 1. Nucleostemin (brown) stains nucleoli in the nuclei (green) of cells in the embryonic rat brain.
Inset: double staining of nucleostemin (red) with DNA dye (blue).
Interestingly, nucleostemin was found to interact with p53 both in pull-down experiments and in co-immunoprecipitation experiments of endogenous proteins. The authors suggest that this interaction may likely occur in the nucleoplasm, as p53 is not normally found in the nucleolus.
Analysis of nucleostemin deletion mutants in overexpression studies allowed the investigators to determine requirements for subnuclear localization. A nucleostemin mutant protein lacking a basic amino-terminal domain displayed diffuse nuclear staining, demonstrating that this region is essential for nucleolar localization. In contrast, nucleostemin lacking either of two putative GTP binding domains exhibited a predominantly nucleolar localization, indicating that nucleostemin may localize to the nucleolus when unbound from GTP (Fig. 2). Paradoxically, forced overexpression of wild-type nucleostemin and its deletion mutants resulted in cell cycle exit or apoptosis, although the physiological relevance of these biological outcomes is uncertain because such high levels of expression may never occur in normal circumstances.
Figure 2. Nucleostemin may regulate p53 function through shuttling between the nucleolus and nucleoplasm.
a, Nucleostemin accumulates predominantly in the nucleolus and to a lesser extent in the nucleoplasm. An overexpression study with nucleostemin deletion mutants led to the working hypothesis that nucleostemin localizes to the nucleolus in its GTP-unbound state, while it is nucleoplasmic upon GTP binding. b, When expressed (or when preferentially in the nucleoplasm), nucleostemin binds p53 and inhibits its growth-suppressive activity. c, When downregulated, such as in differentiating stem cells or in knockdown experiments (or when titrated in the nucleolus), nucleostemin releases the functional block on p53, leading to growth arrest.
Although it is still not known whether nucleostemin acts as a negative or positive regulator of p53 function, a working hypothesis can be formulated on the basis of the preferential expression of nucleostemin in actively proliferating stem cells and cancer cells, and the results in the knockdown experiments: nucleostemin might interact with p53 and inhibit its growth-suppressive function (Fig. 2). In this scenario, the expression of nucleostemin in stem or cancer cells imposes a block on p53 activity (Fig. 2b), while its downregulation during cell differentiation releases this block—allowing for the stabilization or activation of p53 and the induction of target genes critical for cell cycle exit and differentiation, such as p21CIP (Fig. 2c). The regulation of nucleostemin shuttling between nucleolus and nucleoplasm, possibly via GTP binding (Fig. 2a), may in turn modulate the functional cross talk between p53 and nucleostemin.
These findings raise many questions concerning the patho-physiological role of nucleostemin, its mechanism of action and its regulation. For instance, is nucleostemin an accomplice in tumorigenesis? It would be interesting to establish a causative role for nucleostemin misexpression in cellular transformation and determine at which level this protein antagonizes p53 function. Although forced overexpression of nucleostemin induces growth arrest, it is well known that oncogenes such as Ras or c-Myc, once overexpressed in primary cells, can trigger growth arrest or apoptosis—often depending on an intact p53 pathway7. This calls for an assessment of the transforming ability of nucleostemin in combination with other oncogenic events. The tumor types that misexpress nucleostemin and the mechanism and timing of nucleostemin reactivation in cancer cells are also unknown. Moreover, it remains to be determined whether nucleostemin is required for the integrity of the nucleolus. And what are the mechanisms, if any, that regulate the shuttling of nucleostemin between the nucleolus and the nucleoplasm?
The identification of nucleostemin also has potential therapeutic implications. The presence in the protein of two putative GTP binding domains makes it a very attractive target for high-throughput screenings for small molecules that would mimic or compete for GTP binding. Drugs that would compete for GTP binding to nucleostemin may favor its retention in the nucleolus, in turn reverting the p53 blockade. If indeed nucleostemin turns out to have a key role in promoting cancer cell proliferation, these molecules could be tested for their anti-tumor activity. On the other hand, drugs that mimic GTP binding to nucleostemin may potentiate its localization in the nucleoplasmic compartment and in turn promote its block on p53 function. As expansion of stem cells in culture is a key tool for tissue regeneration, such drugs may facilitate stem cell maintenance and propagation.
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