Yoko Itahana

Tumor Suppressor ARF Degrades B23, a Nucleolar Protein Involved in Ribosome Biogenesis and Cell Proliferation

The tumor suppressor ARF induces a p53-dependent and -independent cell cycle arrest. Unlike the nucleoplasmic MDM2 and p53, ARF localizes in the nucleolus. The role of ARF in the nucleolus, the molecular target, and the mechanism of its p53-independent function remains unclear. Here we show that ARF interacts with B23, a multifunctional nucleolar protein involved in ribosome biogenesis, and promotes its polyubiquitination and degradation. Overexpression of B23 induces a cell cycle arrest in normal fibroblasts, whereas in cells lacking p53 it promotes S phase entry. Conversely, knocking down B23 inhibits the processing of preribosomal RNA and induces cell death. Further, oncogenic Ras induces B23 only in ARF null cells, but not in cells that retain wild-type ARF. Together, our results reveal a molecular mechanism of ARF in regulating ribosome biogenesis and cell proliferation via inhibiting B23, and suggest a nucleolar role of ARF in surveillance of oncogenic insults.

Control of the replicative life span of human fibroblasts by p16 and the polycomb protein Bmi-1

ABSTRACT The polycomb protein Bmi-1 represses the INK4a locus, which encodes the tumor suppressors p16 and p14ARF. Here we report that Bmi-1 is downregulated when WI-38 human fibroblasts undergo replicative senescence, but not quiescence, and extends replicative life span when overexpressed. Life span extension by Bmi-1 required the pRb, but not p53, tumor suppressor protein. Deletion analysis showed that the RING finger and helix-turn-helix domains of Bmi-1 were required for life span extension and suppression of p16. Furthermore, a RING finger deletion mutant exhibited dominant negative activity, inducing p16 and premature senescence. Interestingly, presenescent cultures of some, but not all, human fibroblasts contained growth-arrested cells expressing high levels of p16 and apparently arrested by a p53- and telomere-independent mechanism. Bmi-1 selectively extended the life span of these cultures. Low O2 concentrations had no effect on p16 levels or life span extension by Bmi-1 but reduced expression of the p53 target, p21. We propose that some human fibroblast strains are more sensitive to stress-induced senescence and have both p16-dependent and p53/telomere-dependent pathways of senescence. Our data suggest that Bmi-1 extends the replicative life span of human fibroblasts by suppressing the p16-dependent senescence pathway.

Id-1 as a molecular target in therapy for breast cancer cell invasion and metastasis

Mammary epithelial cells constitutively expressing Id-1 protein are unable to differentiate, acquire the ability to proliferate, and invade the extracellular matrix. In addition, Id-1 is aberrantly over-expressed in aggressive and metastatic breast cancer cells, as well as in human breast tumor biopsies from infiltrating carcinomas, suggesting Id-1 might be an important regulator of breast cancer progression. We show that human metastatic breast cancer cells become significantly less invasive in vitro and less metastatic in vivo when Id-1 is down-regulated by stable transduction with antisense Id-1. Expression of the matrix metalloproteinase MT1-MMP is decreased in proportion to the decrease in Id-1 protein levels, representing a potential mechanism for the reduction of invasiveness. Further, to more accurately recapitulate the biology of and potential therapeutic approaches to tumor metastasis, we targeted Id-1 expression systemically in tumor-bearing mice by using a nonviral approach. We demonstrate significant reduction of both Id-1 and MT1-MMP expressions as well as the metastatic spread of 4T1 breast cancer cells in syngeneic BALB/c mice. In conclusion, our studies have identified Id-1 as a critical regulator of breast cancer progression and suggest the feasibility of developing novel therapeutic approaches to target Id-1 expression to reduce breast cancer metastasis in humans.

Id-1 and Id-2 proteins as molecular markers for human prostate cancer progression.

Purpose: Id proteins are dominant-negative regulators of basic helix-loop-helix transcription factors that control malignant cell behavior in many different tissues. This study aimed to identify the potential role of Id-1 and Id-2 proteins as molecular makers for prostate cancer progression. Experimental Design: Using the technique of immunohistochemistry, we determined Id-1 and Id-2 expression in a panel of 67 human prostate biopsies. We also manipulated Id-1 and Id-2 expression in LNCaP and PC3 prostate cancer cell lines and determined the effects on invasion in vitro, matrix metalloproteinase secretion, and proliferation. Results: Both Id-1 and Id-2 proteins were up-regulated during human prostate cancer progression in vivo and were overexpressed in highly aggressive prostate cancer cells. In vitro, constitutive expression of Id-1, and to a lesser extent Id-2, converted nonaggressive LNCaP prostate cancer cells into more proliferative and invasive cells and increased their secretion of matrix metalloproteinases. Conversely, the down-regulation of Id-2 expression in highly metastatic PC3 cells reduced their growth potential and invasiveness. Conclusions: We propose that both Id-1 and Id-2 proteins control prostate cancer cell phenotypes and could serve as molecular markers of aggressive human prostate cancer.

Id-1, ITF-2, and Id-2 Comprise a Network of Helix-Loop-Helix Proteins That Regulate Mammary Epithelial Cell Proliferation, Differentiation, and Apoptosis

Mammary epithelial cells proliferate, invade the stroma, differentiate, and die in adult mammals by mechanisms that are poorly understood. We found that Id-1, an inhibitor of basic helix-loop-helix transcription factors, regulates mammary epithelial cell growth, differentiation, and invasion in culture. Here, we show that Id-1 is expressed highly during mammary development in virgin mice and during early pregnancy, when proliferation and invasion are high. During mid-pregnancy, Id-1 expression declined to undetectable levels as the epithelium differentiated fully. Surprisingly, Id-1 increased during involution, when the epithelium undergoes extensive apoptosis. To determine whether Id-1 regulates both proliferation and apoptosis, we constitutively expressed Id-1 in mammary epithelial cell cultures. Id-1 stimulated proliferation in sparse cultures but induced apoptosis in dense cultures, which reflect epithelial cell density during early pregnancy and involution, respectively. To understand how Id-1 acts, we screened a yeast two-hybrid library from differentiating mammary epithelial cells and identified ITF-2, a basic helix-loop-helix transcription factor, as an Id-1-interacting protein. Overexpression of ITF-2 significantly reduced Id-1-stimulated proliferation and apoptosis. We show further that, in contrast to Id-1, Id-2 was expressed highly in differentiated mammary epithelial cells in vivo and in culture. In culture, Id-2 antisense transcripts blocked differentiation. Our results suggest that Id-1, ITF-2, and Id-2 comprise a network of interacting molecular switches that govern mammary epithelial cell phenotypes.

Proteolytic Enzymes and Altered Glycosylation Modulate Dystroglycan Function in Carcinoma Cells

Alterations in the basement membrane receptor dystroglycan (DG) are evident in muscular dystrophies and carcinoma cells and characterized by a selective loss or modification of the extracellular α-DG subunit. Defects in posttranslational modifications of DG have been identified in some muscular dystrophies, but the underlying modifications in carcinoma cells have not yet been defined. We reveal here multiple posttranslational modifications that modulate the composition and function of DG in normal epithelial cells and carcinoma cells. We show that α-DG is shed from the cell surface of normal and tumorigenic epithelial cells through a proteolytic mechanism that does not require direct cleavage of either α- or β-DG. Shedding is dependent on metalloprotease activity and the proprotein convertase furin. Surprisingly, furin is also found to directly process α-DG as a proprotein substrate, changing the existing model of DG composition. We also show that the glycosylation of α-DG is altered in invasive carcinoma cells, and this modification causes complete loss of laminin binding properties. Together, these data elucidate several novel events regulating the functional composition of DG and reveal defects that arise during cancer progression, providing direction for efforts to restore this link with the basement membrane in carcinoma cells.

Nucleocytoplasmic Shuttling Modulates Activity and Ubiquitination-Dependent Turnover of SUMO-Specific Protease 2

ABSTRACT Small ubiquitin-related modifier (SUMO) proteins are conjugated to numerous polypeptides in cells, and attachment of SUMO plays important roles in regulating the activity, stability, and subcellular localization of modified proteins. SUMO modification of proteins is a dynamic and reversible process. A family of SUMO-specific proteases catalyzes the deconjugation of SUMO-modified proteins. Members of the Sentrin (also known as SUMO)-specific protease (SENP) family have been characterized with unique subcellular localizations. However, little is known about the functional significance of or the regulatory mechanism derived from the specific localizations of the SENPs. Here we identify a bipartite nuclear localization signal (NLS) and a CRM1-dependent nuclear export signal (NES) in the SUMO protease SENP2. Both the NLS and the NES are located in the nonhomologous domains of SENP2 and are not conserved among other members of the SENP family. Using a series of SENP2 mutants and a heterokaryon assay, we demonstrate that SENP2 shuttles between the nucleus and the cytoplasm and that the shuttling is blocked by mutations in the NES or by treating cells with leptomycin B. We show that SENP2 can be polyubiquitinated in vivo and degraded through proteolysis. Restricting SENP2 in the nucleus by mutations in the NES impairs its polyubiquitination, whereas a cytoplasm-localized SENP2 made by introducing mutations in the NLS can be efficiently polyubiquitinated, suggesting that SENP2 is ubiquitinated in the cytoplasm. Finally, treating cells with MG132 leads to accumulation of polyubiquitinated SENP2, indicating that SENP2 is degraded through the 26S proteolysis pathway. Thus, the function of SENP2 is regulated by both nucleocytoplasmic shuttling and polyubiquitin-mediated degradation.

Constitutive expression of the Id-1 promoter in human metastatic breast cancer cells is linked with the loss of NF-1/Rb/HDAC-1 transcription repressor complex

The helix–loop–helix protein Id-1 is a dominant negative regulator of basic helix–loop–helix transcription factors, and plays a key role in the control of breast epithelial cell growth, invasion and differentiation. Previous investigations in our laboratory have shown that Id-1 mRNA was constitutively expressed in highly aggressive and invasive human breast cancer cells in comparison to non-transformed or non-aggressive cancerous cells, and that this loss of regulation is mediated by a 2.2-kb region of the human Id-1 promoter. Here we show that a 31 bp sequence within this 2.2-kb promoter, located 200 bp upstream of the initiation of transcription, is responsible for the constitutive expression of Id-1 in metastatic human breast cancer cells. Using gel shift experiments, we identified a high molecular weight complex present only in non-aggressive breast cancer cells cultured in serum-free medium and which appear to be necessary for proper Id-1 repression. In contrast, nuclear extracts from highly aggressive and metastatic cell lines do not contain this large molecular weight complex. Using DNA affinity precipitation assays (DAPA), we show that this complex contains SP-1, NF-1, Rb and HDAC-1 proteins. On the basis of these findings, we propose a mechanism for the loss of regulation of Id-1 promoter in invasive and metastatic human breast cancer cells.

TP53 Genomic Status Regulates Sensitivity of Gastric Cancer Cells to the Histone Methylation Inhibitor 3-Deazaneplanocin A (DZNep)

Purpose: DZNep (3-deazaneplanocin A) depletes EZH2, a critical component of polycomb repressive complex 2 (PRC2), which is frequently deregulated in cancer. Despite exhibiting promising anticancer activity, the specific genetic determinants underlying DZNep responsiveness in cancer cells remain largely unknown. We sought to determine molecular factors influencing DZNep response in gastric cancer. Experimental Design: Phenotypic effects of DZNep were evaluated in a panel of gastric cancer cell lines. Sensitive lines were molecularly interrogated to identify potential predictors of DZNep responsiveness. The functional importance of candidate predictors was evaluated using short hairpin RNA (shRNA) and siRNA technologies. Results: DZNep depleted PRC2 pathway components in almost all gastric cancer lines, however, only a subset of lines exhibited growth inhibition upon treatment. TP53 genomic status was significantly associated with DZNep cellular responsiveness, with TP53 wild-type (WT) lines being more sensitive (P < 0.001). In TP53-WT lines, DZNep stabilized p53 by reducing ubiquitin conjugation through USP10 upregulation, resulting in activation of canonical p53 target genes. TP53 knockdown in TP53-WT lines attenuated DZNep sensitivity and p53 target activation, showing the functional importance of an intact p53 pathway in regulating DZNep cellular sensitivity. In primary human gastric cancers, EZH2 expression was negatively correlated with p53 pathway activation, suggesting that higher levels of EZH2 may repress p53 activity. Conclusion: Our results highlight an important role for TP53 genomic status in influencing DZNep response in gastric cancer. Clinical trials evaluating EZH2-targeting agents such as DZNep should consider stratifying patients with gastric cancer by their TP53 genomic status. Clin Cancer Res; 18(15); 4201–12. ©2012 AACR.

p53 Oligomerization Is Essential for Its C-terminal Lysine Acetylation

Acetylation of multiple lysine residues in the p53 plays critical roles in the protein stability and transcriptional activity of p53. To better understand how p53 acetylation is regulated, we generated a number of p53 mutants and examined acetylation of each mutant in transfected cells. We found that p53 mutants that are defective in tetramer formation are also defective in C-terminal lysine residue acetylation. Consistently, we found that several cancer-derived p53 mutants that bear mutations in the tetramerization domain cannot form oligomers and are defective in C-terminal lysine acetylation, and these mutants are inactive in p21 transactivation. We demonstrated that the acetyltransferase p300 interacts with and promotes acetylation of wild-type p53 but not with any of the artificially generated or human cancer-derived p53 mutants that are defective in oligomerization. These results, combined with a computer-aided crystal structure analysis, suggest a model in which p53 oligomerization precedes its acetylation by providing docking sites for acetyltransferases.