p27Kip1, a double-edged sword in Shh-mediated medulloblastoma

Medulloblastoma, a brain tumor arising in the cerebellum, is the most common solid childhood malignancy. The current standard of care for medulloblastoma leaves survivors with life-long side effects. Gaining insight into mechanisms regulating transformation of medulloblastoma cells-of-origin may lead to development of better treatments for these tumors. Cerebellar granule neuron precursors (CGNPs) are proposed cells-of-origin for certain classes of medulloblastoma, specifically those marked by aberrant Sonic hedgehog (Shh) signaling pathway activation. CGNPs require signaling by Shh for proliferation during brain development. In mitogen-stimulated cells, nuclear localized cyclin dependent kinase (cdk) inhibitor p27 (Kip1) functions as a checkpoint control at the G1- to S-phase transition by inhibiting cdk2. Recent studies have suggested cytoplasmically localized p27^kip1 acquires oncogenic functions. Here, we show that p27^Kip1 is cytoplasmically localized in CGNPs and mouse Shh-mediated medulloblastomas. Tranasgenic mice bearing an activating mutation in the Shh pathway and lacking one or both p27^Kip1 alleles have accelerated tumor incidence compared to mice bearing both p27^Kip1 alleles. Interestingly, mice heterozygous for p27^Kip1 have decreased survival latency compared to p27^Kip1-null animals. Our data indicate that this may reflect the requirement for at least one copy of p27^Kip1 for recruiting cyclin D/cdk4/6 to promote cell cycle progression yet insufficient expression in the heterozygous or null state to inhibit cyclin E/cdk2. Finally, we find that mis-localized p27^Kip1 may play a positive role in motility in medulloblastoma cells. Together, our data indicate that the dosage of p27^Kip1 plays a role in cell cycle progression and tumor suppression in Shh-mediated medulloblastoma expansion.     

Nmyc upregulation by sonic hedgehog signaling promotes proliferation in developing cerebellar granule neuron precursors

Hedgehog pathway activation is required for expansion of specific neuronal precursor populations during development and is etiologic in the human cerebellar tumor, medulloblastoma. We report that sonic hedgehog (Shh) signaling upregulates expression of the proto-oncogene Nmyc in cultured cerebellar granule neuron precursors (CGNPs) in the absence of new protein synthesis. The temporal-spatial expression pattern of Nmyc, but not other Myc family members, precisely coincides with regions of hedgehog proliferative activity in the developing cerebellum and is observed in medulloblastomas of Patched (Ptch) heterozygous mice. Overexpression of Nmyc promotes cell-autonomous G(1) cyclin upregulation and CGNP proliferation independent of Shh signaling. Furthermore, Myc antagonism in vitro significantly decreases proliferative effects of Shh in cultured CGNPs. Together, these findings identify Nmyc as a direct target of the Shh pathway that functions to regulate cell cycle progression in cerebellar granule neuron precursors.     

β-Arrestin-1 links mitogenic sonic hedgehog signaling to the cell cycle exit machinery in neural precursors

Development of the cerebellum, a brain region regulating posture and coordination, occurs post-natally and is marked by rapid proliferation of granule neuron precursors (CGNPs), stimulated by mitogenic Sonic hedgehog (Shh) signaling. β-Arrestin (βArr) proteins play important roles downstream of Smoothened, the Shh signal transducer. However, whether Shh regulates βArrs and what role it plays in Shh-driven CGNP proliferation remains to be determined. Here, we report that Shh induces βArr1 accumulation and localization to the nucleus, where it participates in enhancing expression of the cyclin dependent kinase (cdk) inhibitor p27, whose accumulation eventually drives CGNP cell cycle exit. βArr1 knockdown enhances CGNP proliferation and reduces p27 expression. Thus, Shh-mediated βArr1 induction represents a novel negative feedback loop within the Shh mitogenic pathway, such that ongoing Shh signaling, while required for CGNPs to proliferate, also sets up a cell-intrinsic clock programming their ultimate exit from the cell cycle.Key words: sonic hedgehog, cerebellum, neural precursor, β-arrestin 1, p27, differentiation     

β-Arrestin-1 links mitogenic sonic hedgehog signaling to the cell cycle exit machinery in neural precursors

Development of the cerebellum, a brain region regulating posture and coordination, occurs post-natally and is marked by rapid proliferation of granule neuron precursors (CGNPs), stimulated by mitogenic Sonic hedgehog (Shh) signaling. β-Arrestin (βArr) proteins play important roles downstream of Smoothened, the Shh signal transducer. However, whether Shh regulates βArrs and what role they play in Shh-driven CGNP proliferation remains to be determined. Here, we report that Shh induces βArr1 accumulation and localization to the nucleus, where it participates in enhancing expression of the cyclin dependent kinase (cdk) inhibitor p27, whose accumulation eventually drives CGNP cell cycle exit. βArr1 knock-down enhances CGNP proliferation and reduces p27 expression. Thus, Shh-mediated βArr1 induction represents a novel negative feedback loop within the Shh mitogenic pathway, such that ongoing Shh signaling, while required for CGNPs to proliferate, also sets up a cell-intrinsic clock programming their ultimate exit from the cell cycle.     

Retinoic Acid-Mediated G1 Arrest Is Associated with Induction of p27 and Inhibition of Cyclin-Dependent Kinase 3 in Human Lung Squamous Carcinoma CH27 Cells

Retinoids are promising agents for the prevention and treatment of several human malignancies including lung cancer. In this study, the effect of retinoic acid (RA) on cell growth and the mechanism of growth modulation were examined in human lung squamous carcinoma CH27 cells. Here we report that RA mediated the dose- and time-dependent growth arrest in G1 phase, accompanied by the up-regulation of p27^Kip1 and the down-regulation of the cyclin-dependent kinase 3 (Cdk3) and p21^CIP1/Waf1 proteins. Furthermore, RA-induced growth arrest of CH27 cells was also associated with increased retinoic acid receptor β (RARβ) and reduced c-Myc expression. However, RA had no effect on the levels of cyclins A, D1, D3, E, or H, or on Cdk2, Cdk4, Cdk5, CDk6, Cdk7, p16^Ink4A, p15^Ink4B, p53, or pRb proteins in CH27 cells. Evaluation of the kinase activity of cyclin–Cdk complexes showed that RA increases p27^Kip1 expression in CH27 cells leading to markedly reduced cyclin A/Cdk2 kinase activity and slightly reduced cyclin E/Cdk2 kinase activity, with no effect on cyclin D/Cdk4 and cyclin D/Cdk6 activities. Moreover, coincident with the decrease in kinase activity was a drastic increase in cyclin A-bound p27^Kip1. These results suggest that increases in the levels of p27^Kip1 and its binding to cyclin A, as well as reduction of Cdk3 protein expression, are strong candidates for the cell cycle regulator that prevents the entry into the S phase in RA-treated CH27 cells, with prolongation of G1 phase and inhibition of DNA synthesis.     

p27Kip1 is required to maintain proliferative quiescence in the adult cochlea and pituitary

Cell cycle inhibitors, such as the cyclin-dependent kinase (Cdk) inhibitor proteins and retinoblastoma (Rb) family members, control exit from the cell cycle during the development of a variety of terminally differentiated tissues. It is unclear whether sustained expression of these proteins is required to prevent cell cycle re-entry in quiescent and terminally differentiated cells. The organ of Corti (cochlear sensory epithelium) and pars intermedia (intermediate lobe of the pituitary) are two tissues that share the characteristic of ongoing cell division in mice lacking either the p27^Kip1 Cdk inhibitor, Ink4 proteins or Rb. Here, we use tamoxifen-inducible mouse models to delete p27^Kip1 in postnatal animals and show this is sufficient to induce proliferation in both the organ of Corti and pars intermedia. Thus, these tissues remain sensitive to the presence of p27^Kip1 even after their developmental exit from the cell cycle. The neonatal cochlea displayed heightened sensitivity to changes in p27^Kip1 expression, with a proliferative response higher than that of constitutive null mice. In adults, the proliferative response was reduced but was accompanied by increased cell survival. In contrast, re-establishment of normal p27^Kip1 expression in animals with established pituitary tumors, in an inducible “knock-on” model, led to cessation of pituitary tumor growth, indicating the cells had maintained their susceptibility to p27-mediated growth suppression. Although restoration of p27^Kip1 did not induce apoptosis, it did lead to resolution of pathological features and normalization of gene expression. Our data underscore the importance of p27^Kip1 expression in the maintenance of cellular quiescence and terminal differentiation.Key words: proliferation, cell cycle, p27, Cdk inhibitor, auditory, cochlea, pituitary     

Genetic Characterization of the Role of the Cip/Kip Family of Proteins as Cyclin-Dependent Kinase Inhibitors and Assembly Factors

The Cip/Kip family, namely, p21^Cip1, p27^Kip1, and p57^Kip2, are stoichiometric cyclin-dependent kinase inhibitors (CKIs). Paradoxically, they have been proposed to also act as positive regulators of Cdk4/6-cyclin D by stabilizing these heterodimers. Loss of p21^Cip1 and p27^Kip1 reduces Cdk4/6-cyclin D complexes, although with limited phenotypic consequences compared to the embryonic lethality of Cdk4/6 or triple cyclin D deficiency. This milder phenotype was attributed to Cdk2 compensatory mechanisms. To address this controversy using a genetic approach, we generated Cdk2^−/− p21^−/− p27^−/− mice. Triple-knockout mouse embryonic fibroblasts (MEFs) displayed minimal levels of D-type cyclins and Cdk4/6-cyclin D complexes. p57^Kip2 downregulation in the absence of p21^Cip1 and p27^Kip1 aggravated this phenotype, yet MEFs lacking all Cip/Kip proteins exhibited increased retinoblastoma phosphorylation, together with enhanced proliferation and transformation capacity. In vivo, Cdk2 ablation induced partial perinatal lethality in p21^−/− p27^−/− mice, suggesting partial Cdk2-dependent compensation. However, Cdk2^−/− p21^−/− p27^−/− survivors displayed all phenotypes described for p27^−/− mice, including organomegalia and pituitary tumors. Thus, Cip/Kip deficiency does not impair interphasic Cdk activity even in the absence of Cdk2, suggesting that their Cdk-cyclin assembly function is dispensable for homeostatic control in most cell types.     

CDK inhibitors,p21 and p27, participate in cell cycle exit of mammalian cardiomyocytes

Mammalian cardiomyocytes actively proliferate during embryonic stages, following which cardiomyocytes exit their cell cycle after birth. The irreversible cell cycle exit inhibits cardiac regeneration by the proliferation of pre-existing cardiomyocytes. Exactly how the cell cycle exit occurs remains largely unknown. Previously, we showed that cyclin E- and cyclin A-CDK activities are inhibited before the CDKs levels decrease in postnatal stages. This result suggests that factors such as CDK inhibitors (CKIs) inhibit CDK activities, and contribute to the cell cycle exit. In the present study, we focused on a Cip/Kip family, which can inhibit cyclin E- and cyclin A-CDK activities. Expression of p21^Cip1 and p27^Kip1 but not p57^Kip2 showed a peak around postnatal day 5, when cyclin E- and cyclin A-CDK activities start to decrease. p21^Cip1 and p27^Kip1 bound to cyclin E, cyclin A and CDK2 at postnatal stages. Cell cycle distribution patterns of postnatal cardiomyocytes in p21^Cip1 and p27^Kip1 knockout mice showed failure in the cell cycle exit at G1-phase, and endoreplication. These results indicate that p21^Cip1 and p27^Kip play important roles in the cell cycle exit of postnatal cardiomyocytes.     

p27Kip1 is required to maintain proliferative quiescence in the adult cochlea and pituitary

Cell cycle inhibitors, such as the cyclin-dependent kinase (Cdk) inhibitor proteins and retinoblastoma (Rb) family members, control exit from the cell cycle during the development of a variety of terminally differentiated tissues. It is unclear whether sustained expression of these proteins is required to prevent cell cycle re-entry in quiescent and terminally differentiated cells. The organ of Corti (cochlear sensory epithelium) and pars intermedia (intermediate lobe of the pituitary) are two tissues that share the characteristic of ongoing cell division in mice lacking either the p27^Kip1 Cdk inhibitor, Ink4 proteins, or Rb. Here, we use tamoxifen-inducible mouse models to delete p27^Kip1 in postnatal animals and show this is sufficient to induce proliferation in both the organ of Corti and pars intermedia. Thus, these tissues remain sensitive to the presence of p27^Kip1 even after their developmental exit from the cell cycle. The neonatal cochlea displayed heightened sensitivity to changes in p27^Kip1 expression, with a proliferative response higher than that of constitutive null mice. In adults, the proliferative response was reduced but was accompanied by increased cell survival. In contrast, re-establishment of normal p27^Kip1 expression in animals with established pituitary tumors, in an inducible “knock-on” model, led to cessation of pituitary tumor growth, indicating the cells had maintained their susceptibility to p27-mediated growth suppression. Although restoration of p27^Kip1 did not induce apoptosis, it did lead to resolution of pathological features and normalization of gene expression. Our data underscore the importance of p27^Kip1 expression in the maintenance of cellular quiescence and terminal differentiation.     

Hyperoxia Induces S-Phase Cell-Cycle Arrest and p21-Independent Cdk2 Inhibition in Human Carcinoma T47D-H3 Cells

Little is known about cell-cycle checkpoint activation by oxidative stress in mammalian cells. The effects of hyperoxia on cell-cycle progression were investigated in asynchronous human T47D-H3 cells, which contain mutated p53 and fail to arrest at G1/S in response to DNA damage. Hyperoxic exposure (95% O₂, 40–64 h) induced an S-phase arrest associated with acute inhibition of Cdk2 activity and DNA synthesis. In contrast, exit from G2/M was not inhibited in these cells. After 40 h of hyperoxia, these effects were partially reversible during recovery under normoxic conditions. The inhibition of Cdk2 activity was not due to degradation of Cdk2, cyclin E or A, nor impairment of Cdk2 complex formation with cyclin A or E and p21^Cip1. The loss of Cdk2 activity occurred in the absence of induction and recruitment of cdk inhibitor p21^Cip1 or p27^Kip1 in cyclin A/Cdk2 or cyclin E/Cdk2 complexes. In contrast, Cdk2 inhibition was associated with increased Cdk2-Tyr15 phosphorylation, increased E2F-1 recruitment, and decreased PCNA contents in Cdk2 complexes. The latter results indicate a p21^Cip1/p27^Kip1-independent mechanism of S-phase checkpoint activation in the hyperoxic T47D cell model investigated.     

Cyclin E2, a Novel G1 Cyclin That Binds Cdk2 and Is Aberrantly Expressed in Human Cancers

A novel cyclin gene was discovered by searching an expressed sequence tag database with a cyclin box profile. The human cyclin E2 gene encodes a 404-amino-acid protein that is most closely related to cyclin E. Cyclin E2 associates with Cdk2 in a functional kinase complex that is inhibited by both p27^Kip1 and p21^Cip1. The catalytic activity associated with cyclin E2 complexes is cell cycle regulated and peaks at the G₁/S transition. Overexpression of cyclin E2 in mammalian cells accelerates G₁, demonstrating that cyclin E2 may be rate limiting for G₁ progression. Unlike cyclin E1, which is expressed in most proliferating normal and tumor cells, cyclin E2 levels were low to undetectable in nontransformed cells and increased significantly in tumor-derived cells. The discovery of a novel second cyclin E family member suggests that multiple unique cyclin E-CDK complexes regulate cell cycle progression.     

Skp2 regulates G2/M progression in a p53-dependent manner

Targeted proteasomal degradation mediated by E3 ubiquitin ligases controls cell cycle progression, and alterations in their activities likely contribute to malignant cell proliferation. S phase kinase-associated protein 2 (Skp2) is the F-box component of an E3 ubiquitin ligase complex that targets p27^Kip1 and cyclin E1 to the proteasome. In human melanoma, Skp2 is highly expressed, regulated by mutant B-RAF, and required for cell growth. We show that Skp2 depletion in melanoma cells resulted in a tetraploid cell cycle arrest. Surprisingly, co-knockdown of p27^Kip1 or cyclin E1 failed to prevent the tetraploid arrest induced by Skp2 knockdown. Enhanced Aurora A phosphorylation and repression of G2/M regulators cyclin B1, cyclin-dependent kinase 1, and cyclin A indicated a G2/early M phase arrest in Skp2-depleted cells. Furthermore, expression of nuclear localized cyclin B1 prevented tetraploid accumulation after Skp2 knockdown. The p53 status is most frequently wild type in melanoma, and the tetraploid arrest and down-regulation of G2/M regulatory genes were strongly dependent on wild-type p53 expression. In mutant p53 melanoma lines, Skp2 depletion did not induce cell cycle arrest despite up-regulation of p27^Kip1. These data indicate that elevated Skp2 expression may overcome p53-dependent cell cycle checkpoints in melanoma cells and highlight Skp2 actions that are independent of p27^Kip1 degradation.     

Requirement of Cyclin E-Cdk2 Inhibition in p16INK4a-Mediated Growth Suppression

Loss-of-function mutations of p16^INK4a have been identified in a large number of human tumors. An established biochemical function of p16 is its ability to specifically inhibit cyclin D-dependent kinases in vitro, and this inhibition is believed to be the cause of the p16-mediated G₁ cell cycle arrest after reintroduction of p16 into p16-deficient tumor cells. However, a mutant of Cdk4, Cdk4^N158, designed to specifically inhibit cyclin D-dependent kinases through dominant negative interference, was unable to arrest the cell cycle of the same cells (S. van den Heuvel and E. Harlow, Science 262:2050–2054, 1993). In this study, we determined functional differences between p16 and Cdk4^N158. We show that p16 and Cdk4^N158 inhibit the kinase activity of cellular cyclin D1 complexes through different mechanisms. p16 dissociated cyclin D1-Cdk4 complexes with the release of bound p27^KIP1, while Cdk4^N158 formed complexes with cyclin D1 and p27. In cells induced to overexpress p16, a higher portion of cellular p27 formed complexes with cyclin E-Cdk2, and Cdk2-associated kinase activities were correspondingly inhibited. Cells engineered to express moderately elevated levels of cyclin E became resistant to p16-mediated growth suppression. These results demonstrate that inhibition of cyclin D-dependent kinase activity may not be sufficient to cause G₁ arrest in actively proliferating tumor cells. Inhibition of cyclin E-dependent kinases is required in p16-mediated growth suppression.     

Phosphorylation of p27Kip1 at Thr187 by Cyclin-dependent Kinase 5 Modulates Neural Stem Cell Differentiation

Cyclin-dependent kinase 5 (Cdk5) plays a key role in the development of the mammalian nervous system; it phosphorylates a number of targeted proteins involved in neuronal migration during development to synaptic activity in the mature nervous system. Its role in the initial stages of neuronal commitment and differentiation of neural stem cells (NSCs), however, is poorly understood. In this study, we show that Cdk5 phosphorylation of p27^Kip1 at Thr187 is crucial to neural differentiation because 1) neurogenesis is specifically suppressed by transfection of p27^Kip1 siRNA into Cdk5^+/+ NSCs; 2) reduced neuronal differentiation in Cdk5^−/− compared with Cdk5^+/+ NSCs; 3) Cdk5^+/+ NSCs, whose differentiation is inhibited by a nonphosphorylatable mutant, p27/Thr187A, are rescued by cotransfection of a phosphorylation-mimicking mutant, p27/Thr187D; and 4) transfection of mutant p27^Kip1 (p27/187A) into Cdk5^+/+ NSCs inhibits differentiation. These data suggest that Cdk5 regulates the neural differentiation of NSCs by phosphorylation of p27^Kip1 at theThr187 site. Additional experiments exploring the role of Ser10 phosphorylation by Cdk5 suggest that together with Thr187 phosphorylation, Ser10 phosphorylation by Cdk5 promotes neurite outgrowth as neurons differentiate. Cdk5 phosphorylation of p27^Kip1, a modular molecule, may regulate the progress of neuronal differentiation from cell cycle arrest through differentiation, neurite outgrowth, and migration.     

Cdk2 as a master of S phase entry: fact or fake?

It has long been believed that Cdk2 and its activator cyclin E play essential roles in the progression of the mitotic cell cycle. However, recent studies using knockout mouse models revealed that neither Cdk2 nor cyclin E are essential in vivo. The purpose of this Perspective is to compare both Cdk2 and cyclin E knockout mice models and to discuss potential mechanisms driving the cell cycle in the absence of Cdk2 or cyclin E. Particular emphasis is placed on possible non-catalytic roles of cyclin E, the expression and activity of the second cyclin binding partner of Cdk2, cyclin A, as well as on the expression and degradation of the Cdk2 inhibitor p27Kip1 in the absence of Cdk2.     

Androgen Suppresses the Proliferation of Androgen Receptor-Positive Castration-Resistant Prostate Cancer Cells via Inhibition of Cdk2, CyclinA,and Skp2

The majority of prostate cancer (PCa) patient receiving androgen ablation therapy eventually develop castration-resistant prostate cancer (CRPC). We previously reported that androgen treatment suppresses Skp2 and c-Myc through androgen receptor (AR) and induced G1 cell cycle arrest in androgen-independent LNCaP 104-R2 cells, a late stage CRPC cell line model. However, the mechanism of androgenic regulation of Skp2 in CRPC cells was not fully understood. In this study, we investigated the androgenic regulation of Skp2 in two AR-positive CRPC cell line models, the LNCaP 104-R1 and PC-3^AR Cells. The former one is an early stage androgen-independent LNCaP cells, while the later one is PC-3 cells re-expressing either wild type AR or mutant LNCaP AR. Proliferation of LNCaP 104-R1 and PC-3^AR cells is not dependent on but is suppressed by androgen. We observed in this study that androgen treatment reduced protein expression of Cdk2, Cdk7, Cyclin A, cyclin H, Skp2, c-Myc, and E2F-1; lessened phosphorylation of Thr14, Tyr15, and Thr160 on Cdk2; decreased activity of Cdk2; induced protein level of p27^Kip1; and caused G1 cell cycle arrest in LNCaP 104-R1 cells and PC-3^AR cells. Overexpression of Skp2 protein in LNCaP 104-R1 or PC-3^AR cells partially blocked accumulation of p27^Kip1 and increased Cdk2 activity under androgen treatment, which partially blocked the androgenic suppressive effects on proliferation and cell cycle. Analyzing on-line gene array data of 214 normal and PCa samples indicated that gene expression of Skp2, Cdk2, and cyclin A positively correlates to each other, while Cdk7 negatively correlates to these genes. These observations suggested that androgen suppresses the proliferation of CRPC cells partially through inhibition of Cyclin A, Cdk2, and Skp2.