Role of the cyclin-dependent kinase inhibitor p27(Kip1) in androgen-induced inhibition of CAMA-1 breast cancer cell proliferation

Androgens are known to inhibit the growth of breast cancer cells, but the molecular mechanism of androgen-induced growth inhibition remains unknown. To address this question, we examined functional and quantitative alterations in cell cycle regulators in the E-responsive CAMA-1 breast cancer cell line. We report here that the androgen 5 alpha-dihydrotestosterone inhibits the proliferation of CAMA-1 breast cancer cells. This inhibition of cell proliferation was dose dependent, and maximal inhibition of E2-stimulated proliferation was observed at the concentration of 1 nM 5 alpha-dihydrotestosterone. 5 alpha-Dihydrotestosterone-induced growth arrest was accompanied by an increase in the proportion of cells in the G(1) phase of the cell cycle. Compared with control cells, 5 alpha-dihydrotestosterone-treated cells showed an increase in the relative proportion of hypophosphorylated retinoblastoma protein consistent with G(1) arrest. In CAMA-1 cells, 5 alpha-dihydrotestosterone caused an accumulation of the cyclin-dependent kinase inhibitor p27(Kip1). Cyclin E-cyclin-dependent kinase-2-associated kinase activity was strongly inhibited in 5 alpha-dihydrotestosterone-treated cells, and immunoprecipitation-Western blot analysis showed an increase in the amount of p27(Kip1) associated with cyclin E-cyclin-dependent kinase-2 complexes. These results suggest that inhibition of breast cancer cell growth by androgens may be mediated at least in part by inactivation of the cyclin E-cyclin-dependent kinase-2 complexes by p27(Kip1).     

p27Kip1 inhibits systemic autoimmunity through the control of Treg cell activity and differentiation

Objective

Despite the importance of Treg cells in the maintenance of immunologic tolerance, the mechanisms that control their generation and activity are unknown. Since the cell cycle inhibitor p27^Kip1 (p27) was involved in T cell anergy, we undertook this study to explore its role in both Treg cell processes.

Methods

The development of type II collagen–induced arthritis (CIA) and lupus‐like abnormalities was compared between transgenic mice overexpressing human Bcl‐2 in T cells (BCL2‐TgT mice) and nontransgenic mice that were deficient or not deficient in p27. The contribution of Treg cells to disease evolution was also explored. Finally, the in vitro activity of Treg cells and their differentiation from naive CD4+ cells was compared between these strains of mice.

Results

BCL2‐TgT mice were protected against CIA by a Treg cell–dependent mechanism. In association with this protection, the overexpression of Bcl‐2 in T cells enhanced the differentiation and activity of Treg cells. Both Bcl‐2 effects were independent of its antiapoptotic activity but dependent on its capacity to induce the expression of p27 that augmented the strength of transforming growth factor β (TGFβ) signaling in T cells. Accordingly, down‐modulation of p27 expression in BCL2‐TgT mice promoted CIA. In addition, p27 deficiency in aged C57BL/6 mice reduced the number and activity of Treg cells and induced the development of mild lupus‐like abnormalities.

Conclusion

Our results point to p27 as a critical regulator of Treg cell differentiation and function through the positive modulation of TGFβ signaling strength in T cells.

     

Expression of Skp2, a p27(Kip1) ubiquitin ligase,in malignant lymphoma: correlation with p27(Kip1) and proliferation index

Reduced levels of p27(Kip1) are frequent in human cancers and have been associated with poor prognosis. Skp2, a component of the Skp1-Cul1-F-box protein (SCF) ubiquitin ligase complex, has been implicated in p27(Kip1) degradation. Increased Skp2 levels are seen in some solid tumors and are associated with reduced p27(Kip1). We examined the expression of these proteins using single and double immunolabeling in a large series of lymphomas to determine if alterations in their relative levels are associated with changes in cell proliferation and lymphoma subgroups. We studied the expression of Skp2 in low-grade and aggressive B-cell lymphomas (n = 86) and compared them with p27(Kip1) and the proliferation index (PI). Fifteen hematopoietic cell lines and peripheral blood lymphocytes were studied by Western blot analysis. In reactive tonsils, Skp2 expression was limited to proliferating germinal center and interfollicular cells. Skp2 expression in small lymphocytic lymphomas (SLLs) and follicular lymphomas (FCLs) was low (mean percentage of positive tumor cells, less than 20%) and was inversely correlated (r = -0.67; P <.0001) with p27(Kip1) and positively correlated with the PI (r = 0.82; P <.005). By contrast, whereas most mantle cell lymphomas (MCLs) demonstrated low expression of p27(Kip1) and Skp2, a subset (n = 6) expressed high Skp2 (exceeding 20%) with a high PI (exceeding 50%). Skp2 expression was highest in diffuse large B-cell lymphomas (DLBCLs) (mean, 22%) and correlated with Ki-67 (r = 0.55; P <.005) but not with p27(Kip1). Cytoplasmic Skp2 was seen in a subset of aggressive lymphomas. Our data provide evidence for p27(Kip1) degradative function of Skp2 in low-grade lymphomas. The absence of this relationship in aggressive lymphomas suggests that other factors contribute to deregulation of p27(Kip1) expression in these tumors.     

Expression of phosphorylated p27(Kip1) protein and Jun activation domain-binding protein 1 in human pituitary tumors

The cyclin-dependent kinase inhibitor p27(Kip1) (p27) plays a pivotal role in controlling cell proliferation during development and tumorigenesis. p27 has been implicated in pituitary tumorigenesis in studies of knockout mice and in analyses of human pituitary tumor samples. In this study, we further explored the role of p27 in human pituitary tumors by measuring levels of phosphorylated p27 (P-p27), and also Jun activation domain-binding protein 1 (Jab1), which is thought to facilitate the phosphorylation and degradation of p27, in normal pituitary tissue (n = 21), pituitary adenomas (n = 75), and pituitary carcinomas (n = 10). The amount of p27 protein in corticotroph adenomas and pituitary carcinomas was much lower than that in normal pituitary tissue or other types of pituitary adenoma. Nuclear P-p27 protein levels were significantly decreased in the adenomas, compared with the normals, and were much lower in the carcinomas, compared with either normal pituitary tissue or pituitary adenomas. However, P-p27 levels in corticotroph adenomas were similar to normal pituitary tissue, thus demonstrating a greatly increased ratio of P-p27 to p27 specifically in corticotroph tumors. No difference was found in Jab1 protein levels in either corticotroph tumors or other pituitary adenomas, compared with normal tissue, but there was a small but significant increase in Jab1 levels in carcinomas. Corticotroph and metastatic tumors both showed a significantly higher Ki-67 labeling index than normal pituitary or other types of pituitary adenomas, and in general the Ki-67 labeling index was negatively correlated with p27 nuclear staining. The amount of p27 and Jab1 mRNA was positively correlated in all pituitary samples studied but did not correlate with the changes in immunostaining. Our findings suggest that in corticotroph tumors there is an accentuated phosphorylation of p27 into P-p27, possibly related to increased cyclin E expression, whereas both p27 and P-p27 are subject to increased degradation in pituitary carcinomas. Such variations in phosphorylation may play a role in pituitary tumorigenesis, but modulation of Jab1 is unlikely to be important in the pathogenesis of pituitary adenomas.     

Gene disruption of p27(Kip1) allows cell proliferation in the postnatal and adult organ of corti

Hearing loss is most often the result of hair-cell degeneration due to genetic abnormalities or ototoxic and traumatic insults. In the postembryonic and adult mammalian auditory sensory epithelium, the organ of Corti, no hair-cell regeneration has ever been observed. However, nonmammalian hair-cell epithelia are capable of regenerating sensory hair cells as a consequence of nonsensory supporting-cell proliferation. The supporting cells of the organ of Corti are highly specialized, terminally differentiated cell types that apparently are incapable of proliferation. At the molecular level terminally differentiated cells have been shown to express high levels of cell-cycle inhibitors, in particular, cyclin-dependent kinase inhibitors [Parker, S. B., et al. (1995) Science 267, 1024-1027], which are thought to be responsible for preventing these cells from reentering the cell cycle. Here we report that the cyclin-dependent kinase inhibitor p27(Kip1) is selectively expressed in the supporting-cell population of the organ of Corti. Effects of p27(Kip1)-gene disruption include ongoing cell proliferation in postnatal and adult mouse organ of Corti at time points well after mitosis normally has ceased during embryonic development. This suggests that release from p27(Kip1)-induced cell-cycle arrest is sufficient to allow supporting-cell proliferation to occur. This finding may provide an important pathway for inducing hair-cell regeneration in the mammalian hearing organ.     

Down-regulation of p21WAF1/CIP1 or p27Kip1 abrogates antiestrogen-mediated cell cycle arrest in human breast cancer cells

Estrogens and antiestrogens influence the G(1) phase of the cell cycle. In MCF-7 breast cancer cells, estrogen stimulated cell cycle progression through loss of the kinase inhibitor proteins (KIPs) p27 and p21 and through G(1) cyclin-dependent kinase (cdk) activation. Treatment with antiestrogen drugs, Tamoxifen or ICI 182780, caused cell cycle arrest, with up-regulation of both p21 and p27 levels, an increase in their binding to cyclin E-cdk2, and kinase inhibition. The requirement for these KIPs in the arrests induced by estradiol depletion or by antiestrogens was investigated with antisense. Antisense inhibition of p21 or p27 expression in estradiol-depleted or antiestrogenarrested MCF-7 led to abrogation of cell cycle arrest, with loss of cyclin E-associated KIPs, activation of cyclin E-cdk2, and S phase entrance. These data demonstrate that depletion of either p21 or p27 can mimic estrogen-stimulated cell cycle activation and indicate that both of these KIPs are critical mediators of the therapeutic effects of antiestrogens in breast cancer.     

Decorin inhibits macrophage colony-stimulating factor proliferation of macrophages and enhances cell survival through induction of p27(Kip1) and p21(Waf1)

Decorin is a small proteoglycan that is ubiquitous in the extracellular matrix of mammalian tissues. It has been extensively demonstrated that decorin inhibits tumor cell growth; however, no data have been reported on the effects of decorin in normal cells. Using nontransformed macrophages from bone marrow, results of this study showed that decorin inhibits macrophage colony-stimulating factor (M-CSF)-dependent proliferation by inducing blockage at the G(1) phase of the cell cycle without affecting cell viability. In addition, decorin rescues macrophages from the induction of apoptosis after growth factor withdrawal. Decorin induces the expression of the cdk inhibitors p21(Waf1) and p27(Kip1). Using macrophages from mice where these genes have been disrupted, inhibition of proliferation mediated by decorin is related to p27(Kip1) expression, whereas p21(Waf1) expression is necessary to protect macrophages from apoptosis. Decorin also inhibits M-CSF-dependent expression of MKP-1 and extends the kinetics of ERK activity, which is characteristic when macrophages become activated instead of proliferating. The effect of decorin on macrophages is not due to its interaction with epidermal growth factor or interferon-gamma receptors. Furthermore, decorin increases macrophage adhesion to the extracellular matrix, and this may be partially responsible for the expression of p27(Kip1) and the modification of ERK activity, but not for the increased cell survival.     

Reduced expression levels of the cell-cycle inhibitor p27Kip1 in human pituitary adenomas

The molecular mechanisms leading to increased cellular proliferation rates and, thus, tumor formation in the anterior pituitary gland are poorly understood. The cyclin-dependent kinase inhibitor p27Kip1 is a key molecule regulating the G1 phase of the cell cycle in many cell types. Furthermore, it was shown that p27 knock-out mice develop pro-opiomelanocortin-positive pituitary tumors. In an effort to clarify the role of p27 in the normal and tumorous human pituitary, we studied the expression of p27 by immunohistochemistry, using a highly specific mouse monoclonal anti-human p27 antibody. Normal pituitaries and 54 pituitary adenomas (twelve somatotrope adenomas, nine prolactinomas, twelve corticotrope adenomas, three TSH-producing tumors, six gonadotrope adenomas, six null cell adenomas, and six oncocytomas) were analyzed. p27 expression was determined semiquantitatively with regard to both the percentage of positive cells and the intensity of the staining. Normal human pituitaries showed strong expression of p27 in most nuclei. In contrast, the levels of p27 were reduced in the majority of the tumors analyzed. Twenty-two tumors (six somatotrope adenomas, five prolactinomas, four corticotrope adenomas, two TSH-producing tumors, two gonadotrope adenomas, and three null cell adenomas) were completely p27-negative. In 18 tumors, p27 expression was found in < or = 10% of the cells. In the other ten tumors, 11-80% of the cells were p27-positive. In summary, we were able to demonstrate reduced expression levels of the cell-cycle inhibitor p27 in tumors derived from all pituitary cell types. Our data indicate that p27 may be an important regulator of cellular proliferation in the anterior pituitary, the underexpression of which could play a role in pituitary tumorigenesis.     

Cell cycle exit during terminal erythroid differentiation is associated with accumulation of p27(Kip1) and inactivation of cdk2 kinase

Progression through the mammalian cell cycle is regulated by cyclins, cyclin- dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CKIs). The function of these proteins in the irreversible growth arrest associated with terminally differentiated cells is largely unknown. The function of Cip/Kip proteins p21(Cip1) and p27(Kip1) during erythropoietin-induced terminal differentiation of primary erythroblasts isolated from the spleens of mice infected with the anemia-inducing strain of Friend virus was investigated. Both p21(Cip1) and p27(Kip1) proteins were induced during erythroid differentiation, but only p27(Kip1) associated with the principal G(1) CDKs-cdk4, cdk6, and cdk2. The kinetics of binding of p27(Kip1) to CDK complexes was distinct in that p27(Kip1) associated primarily with cdk4 (and, to a lesser extent, cdk6) early in differentiation, followed by subsequent association with cdk2. Binding of p27(Kip1) to cdk4 had no apparent inhibitory effect on cdk4 kinase activity, whereas inhibition of cdk2 kinase activity was associated with p27(Kip1) binding, accumulation of hypo-phosphorylated retinoblastoma protein, and G(1) growth arrest. Inhibition of cdk4 kinase activity late in differentiation resulted from events other than p27(Kip1) binding or loss of cyclin D from the complex. The data demonstrate that p27(Kip1) differentially regulates the activity of cdk4 and cdk2 during terminal erythroid differentiation and suggests a switching mechanism whereby cdk4 functions to sequester p27(Kip1) until a specified time in differentiation when cdk2 kinase activity is targeted by p27(Kip1) to elicit G(1) growth arrest. Further, the data imply that p21(Cip1) may have a function independent of growth arrest during erythroid differentiation. (Blood. 2000;96:2746-2754)     

Thyroid hormone regulates the cell cycle inhibitor p27Kip1 in postnatal murine Sertoli cells

Thyroid hormone regulates early postnatal Sertoli cell proliferation. Transient neonatal hypothyroidism allows prolonged postnatal Sertoli cell mitogenesis and doubles adult Sertoli cell numbers, testis weight, and sperm production. The mechanism of this effect is unknown. Cell proliferation is stimulated by cyclins and cyclin-dependent kinases and inhibited by cyclin-dependent kinase inhibitors (CDKIs). T(3) regulates the CDKI p27(Kip1) in other cell types, and mice lacking p27(Kip1) have increased testis size. To test the hypothesis that T(3) regulates Sertoli cell mitogenesis by acting through p27(Kip1), we compared expression of p27(Kip1) in Sertoli cells of testes from euthyroid, hypothyroid, and hyperthyroid mice. At postnatal d 5-25, testes were collected and immunostained for p27(Kip1) expression, or Sertoli cells were isolated enzymatically and used for p27(Kip1) Western blotting. p27(Kip1) immunostaining was low in rapidly proliferating 5-d-old Sertoli cells but had increased strongly in nonproliferating 25-d-old Sertoli cells. p27(Kip1) immunostaining was reduced in Sertoli cells from hypothyroid mice compared with euthyroid controls at 10 and 16 d, consistent with increased Sertoli cell proliferation in these mice. Western blotting corroborated the p27(Kip1) immunostaining, and p27(Kip1) expression was greater in Sertoli cells from control compared with hypothyroid mice at postnatal d 10 and 16, but p27(Kip1) expression was comparable by d 25. Hyperthyroidism increased p27(Kip1) immunostaining relative to controls, and Western analysis indicated that Sertoli cells from 10-d-old hyperthyroid mice expressed more p27(Kip1) than control mice. These results indicate that thyroid hormone status affects p27(Kip1) expression in neonatal Sertoli cells, suggesting that T(3) effects on Sertoli cell proliferation may be mediated through this CDKI.     

Cryptic sequence features within the disordered protein p27Kip1 regulate cell cycle signaling

Peptide motifs embedded within intrinsically disordered regions (IDRs) of proteins are often the sites of posttranslational modifications that control cell-signaling pathways. How do IDR sequences modulate the functionalities of motifs? We answer this question using the polyampholytic C-terminal IDR of the cell cycle inhibitory protein p27^Kip1 (p27). Phosphorylation of Thr-187 (T187) within the p27 IDR controls entry into S phase of the cell division cycle. Additionally, the conformational properties of polyampholytic sequences are predicted to be influenced by the linear patterning of oppositely charged residues. Therefore, we designed sequence variants of the p27 IDR to alter charge patterning outside the primary substrate motif containing T187. Computer simulations and biophysical measurements confirm predictions regarding the impact of charge patterning on the global dimensions of IDRs. Through functional studies, we uncover cryptic sequence features within the p27 IDR that influence the efficiency of T187 phosphorylation. Specifically, we find a positive correlation between T187 phosphorylation efficiency and the weighted net charge per residue of an auxiliary motif. We also find that accumulation of positive charges within the auxiliary motif can diminish the efficiency of T187 phosphorylation because this increases the likelihood of long-range intra-IDR interactions that involve both the primary and auxiliary motifs and inhibit their contributions to function. Importantly, our findings suggest that the cryptic sequence features of the WT p27 IDR negatively regulate T187 phosphorylation signaling. Our approaches provide a generalizable strategy for uncovering the influence of sequence contexts on the functionalities of primary motifs in other IDRs.Intrinsically disordered proteins and regions (IDPs/IDRs) serve as scaffolds for short linear motifs, which are highly conserved functional sequence modules that are typically 3- to 10-residues long (1, 2). Motifs often mediate protein–protein interactions and they encompass sites of specific posttranslational modifications that control a range of regulatory functions in cells (3). An example of cellular regulation that is achieved through posttranslational modifications of specific residues in motifs is the control of cell cycle arrest through Tyr and Thr phosphorylation in linear motifs within the cell cycle inhibitor protein p27^Kip1.Progression through the mammalian cell cycle is controlled by the interplay between cyclin-dependent kinases (Cdks), cyclins, and the Cip/Kip family of Cdk inhibitors that includes p27^Kip1—referred to hereafter as p27 (4). In isolation, p27, which is disordered (5), encompasses two functional domains. The N-terminal domain (p27_1–95) folds upon binding to Cdk2/cyclin A, resulting in complete kinase inhibition. Conversely, whereas p27_1–95 folds upon binding to Cdk2/cyclin A, the C-terminal domain (p27_96–198) remains disordered and participates in a phosphorylation/ubiquitination-signaling cascade (6) (Fig. 1). Proliferative signals cause the activation of nonreceptor tyrosine kinases (NRTKs) that phosphorylate p27 within the Cdk2-bound N-terminal domain at Tyr-88 (Y88) (7). Phosphorylation of Y88 leads to partial restoration of the kinase activity of Cdk2 that is achieved without releasing p27 from the complex with Cdk2/cyclin A; this triggers intracomplex phosphorylation of Thr-187 (T187) near the C terminus of p27, creating a phosphodegron site (8). Subsequent recruitment of the E3 ubiquitin ligase, SCF^Skp2 (9, 10), catalyzes polyubiquitination of three lysine-containing motifs within p27_96–198 (11). Polyubiquitinated p27 is then selectively degraded by the 26S proteasome, releasing fully active Cdk2/cyclin A to drive progression of the cell cycle into S phase (7).Open in a separate windowFig. 1.The p27 IDR integrates proliferative signals from NRTKs to release active Cdk2/cyclin A via T187 phosphorylation. Proliferative signals activate NRTKs that phosphorylate p27 at Y88, partially restoring Cdk2 activity (step 1). This allows for intracomplex phosphorylation of p27 at T187 by Cdk2 and creates a phosphodegron (step 2). Recruitment of the SCF^Skp2 E3 ubiquitin ligase leads to ubiquitination of lysine residues within p27-C (step 3) followed by selective degradation of p27 by the proteasome (step 4) and release of the fully active Cdk2/cyclin A. The primary sequence of the p27 IDR is shown with positively and negatively charged residues depicted in blue and red, respectively. The primary substrate motif is highlighted in yellow.T187 is part of the primary substrate motif T₁₈₇-P₁₈₈-K₁₈₉-K₁₉₀. Phosphorylation of T187 is the key signaling step that leads to p27 degradation and full activation of Cdk2/cyclin A, and this signaling reaction is enabled by disorder within p27_96–198 (6). Here, we investigate the impact of changes to sequence-encoded disorder on the conformational properties of p27_96–198 and the efficiency of T187 phosphorylation. This investigation was motivated by recent studies showing that the conformational properties and the degree of disorder of IDPs/IDRs are influenced by sequence attributes such as the net charge per residue (NCPR) and the fraction of charged residues (12–18). Importantly, a majority of IDPs/IDRs, including p27_96–198, are polyampholytes that are enriched in both positively and negatively charged residues (19–21). In polyampholytes, the sequence patterning of oppositely charged residues is predicted to modulate the degree of chain expansion/compaction and the amplitudes of conformational fluctuations (20). Alterations to the linear patterning of oppositely charged residues leads to changes in the sequence-specific NCPR profiles and these changes can be quantified using a single parameter κ (20). In sequences with low κ values, the oppositely charged residues are well mixed. In such sequences, intrachain electrostatic repulsions and attractions are mutually screened and the result is a heterogeneous ensemble of expanded, random coil-like conformations (20). In contrast, in sequences with high κ values, the opposite charges are segregated within the sequence and electrostatic attractions dominate. The result is an ensemble of compact conformations.We used sequence design to uncover the role of charge patterning on the conformations of p27_96–198 and the impact, if any, on the efficiency of T187 phosphorylation. Specifically, we generated distinct variants of p27_96–198 by manipulating the charge patterning of its sequence between residues 100 and 180 while keeping the amino acid composition fixed. The sequence of the primary motif and its immediate sequence context, namely, positions 181–198, were also held fixed in the designed variants. In a motif-centric model, the efficiency of T187 phosphorylation should depend only on the presence or absence of the primary substrate motif and be insensitive to sequence changes within the unconstrained region of p27. However, contrary to a purely motif-centric model, we have uncovered cryptic sequence features within p27_96–198 that regulate T187 phosphorylation. Our findings suggest that the p27_96–198 IDR is not just a passive tether between the primary motifs encompassing Y88 and T187. Instead, the sequence features within p27_96–198 actively regulate the coupling of Y88 and T187 phosphorylation.     

Inhibition of Akt increases p27Kip1 levels and induces cell cycle arrest in anaplastic large cell lymphoma

Anaplastic large cell lymphoma (ALCL) is a highly proliferative neoplasm that frequently carries the t(2;5)(p23;q35) and aberrantly expresses nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). Previously, NPM-ALK had been shown to activate the phosphatidylinositol 3 kinase (PI3K)/Akt pathway. As the cyclin-dependent kinase (CDK) inhibitor p27(Kip1) (p27) is usually not expressed in ALCL, we hypothesized that activated Akt (pAkt) phosphorylates p27 resulting in increased p27 proteolysis and cell cycle progression. Here we demonstrate that inhibition of pAkt activity in ALCL decreases p27 phosphorylation and degradation, resulting in increased p27 levels and cell cycle arrest. Using immunohistochemistry, pAkt was detected in 24 (57%) of 42 ALCL tumors, including 8 (44%) of 18 ALK-positive tumors and 16 (67%) of 24 ALK-negative tumors, and was inversely correlated with p27 levels. The mean percentage of p27-positive tumor cells was 5% in the pAkt-positive group compared with 26% in the pAkt-negative group (P = .0076). These findings implicate that Akt activation promotes cell cycle progression through inactivation of p27 in ALCL.