Nancy Yeh

Interweaving the Cell Cycle Machinery with Cell Differentiation

A comprehensive understanding of the mechanisms coupling cell cycle exit and differentiation is important for both cancer biology and tissue development. Cancer cells can arise from either stem/progenitor cells that fail to exit the cell cycle and differentiate, or from de-differentiated cells that have re-entered the cell cycle. Much of our current understanding of this coupling is based on observations made in transformed cell lines. These studies have shown that enforcing proliferation prevents differentiation and inducing growth arrest leads to differentiation; thus, one widely-held view is that changes in cell cycle regulators simply induces cell cycle exit, a pre-requisite for differentiation. However, recent evidence indicates that cell cycle regulators can affect differentiation in other ways as well. They can have a role establishing the new transcriptional program that accompanies differentiation—in its most radical form, the molecular mechanism of arrest might even be an integral component of the differentiation program. Additionally, the regulators or mechanisms that prevent the re-entry of cells into the proliferative cycle may not be those that induce exit from the cell cycle. Our goal in this perspective is to highlight examples from our laboratory that provided a broader understanding of the types of roles that cell cycle regulators play during differentiation, beginning with the phenotypes observed in mice.

Somatic cell type specific gene transfer reveals a tumor-promoting function for p21Waf1/Cip1

How proteins participate in tumorigenesis can be obscured by their multifunctional nature. For example, depending on the cellular context, the cdk inhibitors can affect cell proliferation, cell motility, apoptosis, receptor tyrosine kinase signaling, and transcription. Thus, to determine how a protein contributes to tumorigenesis, we need to evaluate which functions are required in the developing tumor. Here we demonstrate that the RCAS/TvA system, originally developed to introduce oncogenes into somatic cells of mice, can be adapted to allow us to define the contribution that different functional domains make to tumor development. Studying the development of growth‐factor‐induced oligodendroglioma, we identified a critical role for the Cy elements in p21, and we showed that cyclin D1T286A, which accumulates in the nucleus of p21‐deficient cells and binds to cdk4, could bypass the requirement for p21 during tumor development. These genetic results suggest that p21 acts through the cyclin D1–cdk4 complex to support tumor growth, and establish the utility of using a somatic cell modeling system for defining the contribution proteins make to tumor development.

TRIM3, a tumor suppressor linked to regulation of p21(Waf1/Cip1.).

The TRIM family of genes is largely studied because of their roles in development, differentiation and host cell antiviral defenses; however, roles in cancer biology are emerging. Loss of heterozygosity of the TRIM3 locus in ∼20% of human glioblastomas raised the possibility that this NHL-domain containing member of the TRIM gene family might be a mammalian tumor suppressor. Consistent with this, reducing TRIM3 expression increased the incidence of and accelerated the development of platelet-derived growth factor -induced glioma in mice. Furthermore, TRIM3 can bind to the cdk inhibitor p21WAF1/CIP1. Thus, we conclude that TRIM3 is a tumor suppressor mapping to chromosome 11p15.5 and that it might block tumor growth by sequestering p21 and preventing it from facilitating the accumulation of cyclin D1–cdk4.

Cooperation between p27 and p107 during endochondral ossification suggests a genetic pathway controlled by p27 and p130

ABSTRACT Pocket proteins and cyclin-dependent kinase (CDK) inhibitors negatively regulate cell proliferation and can promote differentiation. However, which members of these gene families, which cell type they interact in, and what they do to promote differentiation in that cell type during mouse development are largely unknown. To identify the cell types in which p107 and p27 interact, we generated compound mutant mice. These mice were null for p107 and had a deletion in p27 that prevented its binding to cyclin-CDK complexes. Although a fraction of these animals survived into adulthood and looked similar to single p27 mutant mice, a larger number of animals died at birth or within a few weeks thereafter. These animals displayed defects in chondrocyte maturation and endochondral bone formation. Proliferation of chondrocytes was increased, and ectopic ossification was observed. Uncommitted mouse embryo fibroblasts could be induced into the chondrocytic lineage ex vivo, but these cells failed to mature normally. These results demonstrate that p27 carries out overlapping functions with p107 in controlling cell cycle exit during chondrocyte maturation. The phenotypic similarities between p107−/−p27D51/D51 and p107−/−p130−/− mice and the cells derived from them suggest that p27 and p130 act in an analogous pathway during chondrocyte maturation.

Tyrosine Phosphorylation of the p21 Cyclin-dependent Kinase Inhibitor Facilitates the Development of Proneural Glioma

Background: Phosphorylation of Tyr in the 310 helix of p27 reduces its inhibitory activity on cyclin-CDK complexes. Results: Mutation of this site to Phe reduces the tumor-promoting activity of p21 in the RCAS-PDGF-HA/nestin-TvA mouse model of proneural gliomagenesis. Conclusion: Tyr phosphorylation of p21 contributes to its oncogenic role. Significance: This mouse model establishes the significance of this modification for the nuclear accumulation of cyclin D1-CDK4. Phosphorylation of Tyr-88/Tyr-89 in the 310 helix of p27 reduces its cyclin-dependent kinase (CDK) inhibitory activity. This modification does not affect the interaction of p27 with cyclin-CDK complexes but does interfere with van der Waals and hydrogen bond contacts between p27 and amino acids in the catalytic cleft of the CDK. Thus, it had been suggested that phosphorylation of this site could switch the tumor-suppressive CDK inhibitory activity to an oncogenic activity. Here, we examined this hypothesis in the RCAS-PDGF-HA/nestin-TvA proneural glioma mouse model, in which p21 facilitates accumulation of nuclear cyclin D1-CDK4 and promotes tumor development. In these tumor cells, approximately one-third of the p21 is phosphorylated at Tyr-76 in the 310 helix. Mutation of this residue to glutamate reduced inhibitory activity in vitro. Mutation of this residue to phenylalanine reduced the tumor-promoting activity of p21 in the animal model, whereas glutamate or alanine substitution allowed tumor formation. Consequently, we conclude that tyrosine phosphorylation contributes to the conversion of CDK inhibitors from tumor-suppressive roles to oncogenic roles.

p27kip1 Protein Levels Reflect a Nexus of Oncogenic Signaling during Cell Transformation

Background: Different oncogenes or a reduction in the activity of the PP2A tumor suppressor substitute for the expression of SV40 small t-antigen in cell transformation. Results: These oncogenes and PP2A converge at p27. Conclusion: p27 levels reflect the signaling milieu controlled by oncogenic activation and PP2A. Significance: Understanding what p27 levels reflect in transformed cells is crucial for understanding its significance as a biomarker. SV40 small t-antigen (ST) collaborates with SV40 large T-antigen (LT) and activated rasv12 to promote transformation in a variety of immortalized human cells. A number of oncogenes or the disruption of the general serine-threonine phosphatase protein phosphatase 2A (PP2A) can replace ST in this paradigm. However, the relationship between these oncogenes and PP2A activity is not clear. To address this, we queried the connectivity of these molecules in silico. We found that p27 was connected to each of those oncogenes that could substitute for ST. We further determined that p27 loss can substitute for the expression of ST during transformation of both rodent and human cells. Conversely, knock-in cells expressing the degradation-resistant S10A and T187A mutants of p27 were resistant to the transforming activities of ST. This suggests that p27 is an important target of the tumor-suppressive effects of PP2A and likely an important target of the multitude of cellular oncoproteins that emulate the transforming function of ST.

Increased number of multi-oocyte follicles (MOFs) in juvenile p27Kip1 mutant mice: potential role of granulosa cells

STUDY QUESTION Why are female mice that lack a functional p27 protein infertile? SUMMARY ANSWER The absence of a functional p27 leads to a dramatic increase in the number of multi-oocyte follicles (MOFs) in juvenile female mice; p27 would promote the individualization of follicles favoring the development of fertile eggs. WHAT IS KNOWN ALREADY p27-/- female mice are infertile. p27 suppresses excessive follicular endowment and activation and promotes follicular atresia in mice. MATERIALS AND METHODS Ovaries from wild type (WT) and p27Kip1 mutant mice aged 2, 4 and 12 weeks were subjected to immunohistochemistry/immunofluorescence. The slides with whole organs serially sectioned were scanned and examined by image analysis. MAIN RESULTS AND THE ROLE OF CHANCE Compared with WT, p27Kip1 mutant pre-pubertal mice had a greater number of oocytes, a greater number of growing follicles and a greater number of MOFs. These differences were statistically significant (P < 0.05), particularly in the case of MOFs (P > 0.001). The unusually large number of MOFs in juvenile p27-deficient mice is a novel observation. In WT mice p27 protein remains present in the oocyte nucleus but gradually decreases in the ooplasm during follicular growth, while granulosa cells show dynamic, follicle stage-related changes. LIMITATIONS, REASONS FOR CAUTION These results have been obtained in mice and they cannot be directly extrapolated to humans. WIDER IMPLICATIONS OF THE FINDINGS The dramatic increase in the numbers of MOFs in juvenile p27 mutants has not been previously reported. The number of MOFs declines sharply as the mice become sexually mature, pointing to their negative selection. These findings open a new approach to the study of sterility. STUDY FUNDING/COMPETING INTERESTS This study has been funded by the Basque Government, Dept. of Health grant 2007111063 and Dept. of Industry (Saiotek) grant S-PC11UN008. Jairo Perez-Sanz was the recipient of a grant from Fundación Jesús de Gangoiti Barrera. The authors have no conflicts of interest to declare.