The cyclin-dependent kinase inhibitors p15INK4b and p57KIP2 are important regulators of the cell cycle, and their abnormal expression has been detected in various tumors. However, little is known about the role of p15INK4b and p57KIP2 in the pathogenesis of vulvar carcinoma, and the prognostic impact is still unknown. In our current study, we examined the expression of p15INK4b and p57KIP2 in a large series of vulvar squamous cell carcinomas to elucidate the prognostic impact.
Expression of p15INK4b and p57KIP2 were examined in 297 vulvar squamous cell carcinomas using immunohistochemistry. Both uni- and multivariate analysis of prognostic factors were performed, and correlations with clinicopathologic parameters were examined.
Compared to the high levels of p15INK4b and p57KIP2 in normal vulvar squamous epithelium, low levels of p15INK4b and p57KIP2 were found in 82% and 44% of vulvar carcinomas, respectively. Low levels of p15INK4b and p57KIP2 correlated significantly with malignant features, including large tumor diameter (p = 0.03 and p = 0.001, respectively) and increased invasiveness (p = 0.003 and p = 0.04, respectively). Although p15INK4b and p57KIP2 levels could not be identified as prognostic markers, combined analysis of p14ARF/p15INK4b/p16INK4a showed that patients whose tumors expressed low levels of two or three of these INK4 proteins had a worse prognosis than those with only low levels of one or no protein (univariate analysis p = 0.02). The independent prognostic significance of these INK4 proteins was confirmed by multivariate analysis (p = 0.008).
We show for the first time that p15INK4b and p57KIP2 may be involved in the progression of vulvar carcinomas and the combined p14ARF/p15INK4b/p16INK4a status was a statistically independent prognostic factor.
Cell cycle progression is regulated by the combined action of cyclins, cyclin-dependent kinases (CDKs), and CDK-inhibitors (CDKi), which are negative cell cycle regulators. p27KIP1 is a CDKi key in cell cycle regulation, whose degradation is required for G1/S transition. In spite of the absence of p27KIP1 expression in proliferating lymphocytes, some aggressive B-cell lymphomas have been reported to show an anomalous p27KIP1 staining. We analysed p27KIP1 expression in a series of Diffuse Large B-cell Lymphoma (DLBCL), correlating it with the proliferative index and clinical outcome, to characterize the implications of this anomalous staining in lymphomagenesis in greater depth. For the above mentioned purposes, an immunohistochemical technique in paraffin-embedded tissues was employed, using commercially available antibodies, in a series of 133 patients with known clinical outcomes. Statistical analysis was performed in order to ascertain which clinical and molecular variables may influence outcome, in terms of disease-free survival (DFS) and overall survival (OS). The relationships between p27KIP1 and MIB-1 (Ki-67) were also tested. An abnormally high expression of p27KIP1 was found in lymphomas of this type. The overall correlation between p27KIP1 and MIB-1 showed there to be no significant relationship between these two parameters, this differing from observations in reactive lymphoid and other tissues. Analysis of the clinical relevance of these findings showed that a high level of p27KIP1 expression in this type of tumour is an adverse prognostic marker, in both univariate and multivariate analysis. These results show that there is abnormal p27KIP1 expression in DLBCL, with adverse clinical significance, suggesting that this anomalous p27KIP1 protein may be rendered non-functional through interaction with other cell cycle regulator proteins. © 1999 Cancer Research Campaign
p27KIP1; MIB1; CDK inhibitors; diffuse large B-cell lymphoma; survival analysis
p27Kip1 is a potent inhibitor of cyclin-dependent kinases that drive G1-to-S cell-cycle transition. Reduced p27Kip1 expression is prevalent in a wide range of human tumours; however, the exact mechanism(s) of p27Kip1-mediated tumour suppression remains obscure. In the present study, we identified a close inverse relationship between p27Kip1 and EGFR (epidermal growth factor receptor) expression: the parental T24 human bladder cancer cells had high p27Kip1 expression but low EGFR expression and, in striking contrast, the metastatic derivative of T24 (T24T) had low p27Kip1 expression but high EGFR expression. This relationship was also found in various human cancer tissues, and was not only just correlative but also causal; depletion of p27Kip1 in MEF (mouse embryonic fibroblast) cells resulted in markedly elevated EGFR expression, a result reproducible with an Egfr promoter-luciferase reporter in both T24 and MEF cells, suggesting transcriptional repression of EGFR by p27Kip1. Indeed, p27Kip1 was found to regulate EGFR expression via the JNK (c-Jun N-terminal kinase)/c-Jun transcription factor: p27Kip1 deficiency activated JNK/c-Jun, whereas inhibition of JNK/c-Jun by dominant-negative mutants dramatically repressed Egfr transcription. Furthermore, the proximal promoter of the Egfr gene was crucial for its transcription, where the recruiting activity of c-Jun was much greater in p27Kip1−/− cells than in p27Kip1+/+ cells. Introduction of GFP–p27Kip1 into T24T cells suppressed JNK/c-Jun activation, EGFR expression and anchorage-independent growth. The results of the present study demonstrate that p27Kip1 suppresses JNK/c-Jun activation and EGFR expression in MEFs and human bladder cancer cells, and the results obtained are consistent with those from human cancer specimens. The present study provides new insights into p27Kip1 suppression of cancer cell growth, migration and metastasis.
An inverse relationship between p27Kip1 and EGFR expression in parental T24 human bladder cancer cells and various human cancer tissues was found. Depletion of p27Kip1 in cells markedly elevated EGFR expression through transcriptional repression of Egfr by p27Kip1 via the JNK/c-Jun cascade.
bladder cancer; c-Jun N-terminal kinase (JNK)/c-Jun pathway; epidermal growth factor receptor (EGFR); p27Kip1; signal transduction pathway; AP-1, activator protein 1; BME, basal medium Eagle; CDK, cyclin-dependent kinase; DMEM, Dulbecco’s modified Eagle’s medium; EGFR, epidermal growth factor receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HSF-1, heat-shock factor 1; Hsp, heat-shock protein; IHC, immunohistochemistry; JNK, c-Jun N-terminal kinase; MEF, mouse embryonic fibroblast; RT, reverse transcription; SP1, specificity protein 1
Janus kinase 2 (JAK2) couples ligand activation of cell surface cytokine receptors to the regulation of cellular functions including cell cycle progression, differentiation and apoptosis. It thereby coordinates biological programs such as development and hematopoiesis. Unscheduled activation of JAK2 by point mutations or chromosomal translocations can induce hyperproliferation and hematological malignancies. Typical signal transduction by the JAK2 tyrosine kinase comprises phosphorylation of STAT transcription factors. In this study, we describe the identification of the cyclin-dependent kinase (CDK) inhibitor p27Kip1 as a novel JAK2 substrate. JAK2 can directly bind and phosphorylate p27Kip1. Both, the JAK2 FERM domain and its kinase domain bind to p27Kip1. JAK2 phosphorylates tyrosine residue 88 (Y88) of p27Kip1. We previously reported that Y88 phosphorylation of p27Kip1 by oncogenic tyrosine kinases impairs p27Kip1-mediated CDK inhibition, and initiates its ubiquitin-dependent proteasomal degradation. Consistently, we now find that active oncogenic JAK2V617F reduces p27Kip1 stability and protein levels in patient-derived cell lines harboring the mutant JAK2V617F allele. Moreover, tyrosine phosphorylation of p27Kip1 is impaired and p27Kip1 expression is restored upon JAK2V617F inactivation by small hairpin RNA-mediated knockdown or by the pyridone-containing tetracycle JAK inhibitor-I, indicating that direct phosphorylation of p27Kip1 can contribute to hyperproliferation of JAK2V617F-transformed cells. Activation of endogenous JAK2 by interleukin-3 (IL-3) induces Y88 phosphorylation of p27Kip1, thus unveiling a novel link between cytokine signaling and cell cycle control in non-transformed cells. Oncogenic tyrosine kinases could use this novel pathway to promote hyperproliferation in tumor cells.
cell cycle control; CDK inhibitors; p27Kip1; tyrosine kinases; JAK2; JAK2V617F
p27Kip1 is an inhibitor of the cyclin-dependent kinases and it plays an inhibitory role in the progression of cell cycle through G1 phase. To investigate the mechanism of cell cycle inhibition by p27Kip1, we constructed a cell line that inducibly expresses p27Kip1 upon addition of isopropyl-1-thio-beta-D-galactopyranoside in the culture medium. Isopropyl-1-thio-beta-D-galactopyranoside-induced expression of p27Kip1 in these cells causes a specific reduction in the expression of the E2F-regulated genes such as cyclin E, cyclin A, and dihydrofolate reductase. The reduction in the expression of these genes correlates with the p27Kip1-induced accumulation of the repressor complexes of the E2F family of factors (E2Fs). Our previous studies indicated that p21WAF1 could disrupt the interaction between cyclin/cyclin-dependent kinase 2 (cdk2) and the E2F repressor complexes E2F-p130 and E2F-p107. We show that p27Kip1, like p21WAF1, disrupts cyclin/cdk2-containing complexes of E2F-p130 leading to the accumulation of the E2F-p130 complexes, which is found in growth-arrested cells. In transient transfection assays, expression of p27Kip1 specifically inhibits transcription of a promoter containing E2F-binding sites. Mutants of p27Kip1 harboring changes in the cyclin- and cdk2-binding motifs are deficient in inhibiting transcription from the E2F sites containing reporter gene. Moreover, these mutants of p27Kip1 are also impaired in disrupting the interaction between cyclin/cdk2 and the repressor complexes of E2Fs. Taken together, these observations suggest that p27Kip1 reduces expression of the E2F-regulated genes by generating repressor complexes of E2Fs. Furthermore, the results also demonstrate that p27Kip1 inhibits expression of cyclin A and cyclin E, which are critical for progression through the G1-S phases.
The cyclin-dependent kinase inhibitor p27Kip1 functions during normal cerebellar development and has demonstrated tumor suppressor functions in mouse models of medulloblastoma. Because P27 loss is associated with increased proliferation, we assessed whether P27 absence in surgical medulloblastoma specimens correlated with response to therapy in pediatric patients enrolled in two large studies. Additionally, we examined the functional consequence of p27Kip1 loss in the SmoA1 medulloblastoma model to distinguish whether p27Kip1 reduces tumor initiation or slows tumor progression.
Analysis of 87 well-characterized patient samples identified a threshold of P27 staining at which significant P27 loss correlated with poor patient outcome. The same criteria, applied to a second test set of tissues from 141 patients showed no difference in survival between patients with minimal P27 staining and others, suggesting that P27 levels alone are not a sufficient prognostic indicator for identifying standard-risk patients that may fail standard therapy. These findings were in contrast to prior experiments completed using a mouse medulloblastoma model. Analysis of cerebellar tumor incidence in compound mutant mice carrying the activated Smoothened (SmoA1) allele that were heterozygous or nullizygous for p27Kip1 revealed that p27Kip1 loss did not alter the frequency of tumor initiation. Tumors haploinsufficient or nullizygous for p27Kip1 were, however, more invasive and displayed a higher proliferative index, suggesting p27Kip1 loss may contribute to SmoA1 medulloblastoma progression.
These studies revealed P27 loss affects medulloblastoma progression rather than initiation and that this putative biomarker should not be used for stratifying children with medulloblastoma to risk-based therapeutic regimens.
Medulloblastoma; p27Kip1; Sonic hedgehog; Cerebellum; Mouse cancer models; Prognostic factors
p27Kip1 plays a major role as a negative regulator of the cell cycle. The regulation of p27Kip1 degradation is mediated by its specific ubiquitin ligase subunits S-phase kinase protein (Skp) 2 and cyclin-dependent kinase subunit (Cks) 1. However, little is known regarding the prognostic utility of p27Kip1, Skp2 and Cks1 expression in renal cell carcinoma.
Immunohistochemistry was performed for p27Kip1, Skp2 and Cks1 in tissue microarrays of 482 renal cell carcinomas with follow-up. The data were correlated with clinicopathological features. The univariate and multivariate survival analyses were also performed to determine their prognostic significance.
Immunoreactivity of p27Kip1, Skp2 and Cks1 was noted in 357, 71 and 82 patients, respectively. Skp2 and Cks1 expression were not noted in chromophobe cancers. A strong correlation was found between Skp2 and Cks1 expression (P < 0.001), both of which were inversely related to p27Kip1 levels (P = 0.006 and P < 0.001), especially in primary and clear-cell cancers. Low p27Kip1 expression and Skp2 expression were correlated with larger tumor size and higher stage, as well as tumor necrosis. Cks1 expression was only correlated with tumor size. In univariate analysis, low p27Kip1 expression, Skp2 and Cks1 expression were all associated with a poor prognosis, while in multivariate analysis, only low p27Kip1 expression were independent prognostic factors for both cancer specific survival and recurrence-free survival in patients with RCC.
Our results suggest that immunohistochemical expression levels of p27Kip1, Skp2 and Cks1 may serve as markers with prognostic value in renal cell carcinoma.
Overexpression of the ErbB2 receptor, a major component of the ErbB receptor signaling network, contributes to the development of a number of human cancers. ErbB2 presents itself, therefore, as a target for antibody-mediated therapies. In this respect, anti-ErbB2 monoclonal antibody 4D5 specifically inhibits the growth of tumor cells overexpressing ErbB2. We have analyzed the effect of 4D5-mediated ErbB2 inhibition on the cell cycle of the breast tumor cell line BT474. 4D5 treatment of BT474 cells resulted in a G1 arrest, preceded by rapid dephosphorylation of ErbB2, inhibition of cytoplasmic signal transduction pathways, accumulation of the cyclin-dependent kinase inhibitor p27Kip1, and inactivation of cyclin-Cdk2 complexes. Time courses demonstrated that 4D5 treatment redirects p27Kip1 onto Cdk2 complexes, an event preceding increased p27Kip1 expression; this correlates with the downregulation of c-Myc and D-type cyclins (proteins involved in p27Kip1 sequestration) and the loss of p27Kip1 from Cdk4 complexes. Similar events were observed in ErbB2-overexpressing SKBR3 cells, which exhibited reduced proliferation in response to 4D5 treatment. Here, p27Kip1 redistribution resulted in partial Cdk2 inactivation, consistent with a G1 accumulation. Moreover, p27Kip1 protein levels remained constant. Antisense-mediated inhibition of p27Kip1 expression in 4D5-treated BT474 cells further demonstrated that in the absence of p27Kip1 accumulation, p27Kip1 redirection onto Cdk2 complexes is sufficient to inactivate Cdk2 and establish the G1 block. These data suggest that ErbB2 overexpression leads to potentiation of cyclin E-Cdk2 activity through regulation of p27Kip1 sequestration proteins, thus deregulating the G1/S transition. Moreover, through comparison with an ErbB2-overexpressing cell line insensitive to 4D5 treatment, we demonstrate the specificity of these cell cycle events and show that ErbB2 overexpression alone is insufficient to determine the cellular response to receptor inhibition.
p57Kip2, a cyclin-dependent kinase inhibitor, is considered to be a candidate tumor suppressor gene that has been implicated in Beckwith-Wiedemann syndrome and sporadic cancers. In addition, decreased expression of p57Kip2 protein has been frequently observed in pancreatic, lung, breast, bladder, gastrointestinal tract and prostate cancers. However, p57Kip2 gene mutations are rare in these cancers suggesting that other unknown mechanisms might be at play in reducing its expression. The aim of this study was to investigate the molecular mechanism of down-regulation of p57Kip2 in prostate cancer.
We observed a significant negative correlation between the expression of p57Kip2 and microRNA-21 (miR-21) in prostate cancer samples and after androgen deprivation with castration in the CWR22 human prostate cancer xenograft model. We report that miR-21 targeted the coding region and decreased p57Kip2 mRNA and protein levels in prostate cancer cells. Conversely, inhibition of endogenous miR-21 by an anti-miR-21 inhibitor strongly induced p57Kip2 expression. Furthermore, we found that knockdown of p57Kip2 reversed the effects of the anti-miR-21 inhibitor on cell migration and anchorage-independent cell growth.
Our results indicate that miR-21 is able to downregulate p57Kip2 expression by targeting the coding region of the gene and is also able to attenuate p57Kip2 mediated functional responses. This is the first report demonstrating that p57Kip2 is a novel target of miR-21 in prostate cancer and revealing a novel oncogenic function of this microRNA.
Electronic supplementary material
The online version of this article (doi:10.1186/1476-4598-13-212) contains supplementary material, which is available to authorized users.
p57Kip2; microRNA-21 and prostate cancer
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 p27Kip1 functions as a checkpoint control at the G1- to S-phase transition by inhibiting Cdk2. Recent studies have suggested that cytoplasmically localized p27Kip1 acquires oncogenic functions. Here, we show that p27Kip1 is cytoplasmically localized in CGNPs and mouse Shh-mediated medulloblastomas. Transgenic mice bearing an activating mutation in the Shh pathway and lacking one or both p27Kip1 alleles have accelerated tumor incidence compared to mice bearing both p27Kip1 alleles. Interestingly, mice heterozygous for p27Kip1 have decreased survival latency compared to p27Kip1-null animals. Our data indicate that this may reflect the requiremen of at least one copy of p27Kip1 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 mislocalized p27Kip1 may play a positive role in motility in medulloblastoma cells. Together, our data indicate that the dosage of p27Kip1 plays a role in cell cycle progression and tumor suppression in Shh-mediated medulloblastoma expansion.
p27; Kip1; medulloblastoma; cerebellum; cell cycle; Sonic hedgehog; tumor; motility; RhoA
We have investigated the regulation of p27kip1, a cyclin-dependent kinase inhibitor, in BALB/c 3T3 cells during growth factor-stimulated transition from quiescence (G0) to a proliferative (G1) state. The level of p27kip1 protein falls dramatically after mitogenic stimulation and is accompanied by a decrease in cyclin E associated p27kip1, as well as a transient increase in cyclin D1-associated p27kip1 that later declines concomitantly with the loss of total p27kip1. Analysis of metabolically labelled cells revealed that cyclin D2, cyclin D3, and cdk4 were also partnered with p27kip1 in quiescent BALB/c 3T3 cells and that this association decreased after platelet-derived growth factor (PDGF) treatment. Furthermore, the decline in p27kip1 and reduced association with cyclin D3, initiated by the addition of PDGF but not plasma-derived factors, suggested that these changes are involved in competence, the first step in the exit from G0. Synthesis of p27kip1 as determined by incorporation of [35S]methionine was repressed upon mitogenic stimulation, and PDGF was sufficient to elicit this repression within 2 to 3 h. Pulse-chase experiments demonstrated the reduced rate of synthesis was not the result of an increased rate of degradation. Full repression of p27kip1 synthesis required the continued presence of PDGF and failed to occur in the presence of the RNA polymerase inhibitor 5,6-dichlorobenzimidazole riboside. These characteristics demonstrate that repression was a late effect of PDGF and was consistent with our finding that conditional expression of activated H-ras did not affect synthesis of p27kip1. Northern (RNA) analysis of p27kip1 mRNA revealed that the repression was not accompanied by a corresponding decrease in p27kip1 mRNA, suggesting that the PDGF-regulated decrease in p27kip1 expression occurred through a translational mechanism.
Nuclear expression of the cell cycle inhibitor p27KIP1 is reduced in a variety of human malignancies, including breast cancer. Loss of nuclear p27KIP1 during tumor progression, documented by immunohistochemistry (IHC), has been studied for its potential prognostic implication. We examined by IHC the association between nuclear p27KIP1 expression and hormone receptor status in T1N0M0 breast cancer. Patients and Methods: The correlation between nuclear p27KIP1 expression and estrogen (ER) and progesterone (PR) hormone receptor status was analyzed in 122 human T1N0M0 (68 T1a/b, 54 T1c) breast cancer specimens. All patients were staged as N0 by axillary node dissection. Results: A statistically significant reduction in p27KIP1 expression was observed as tumor size increased from T1a/b (7%) to T1c (22%). The proportion of tumors with low nuclear p27KIP1 expression was higher in the ER-negative/PR-negative group compared to the ER-positive/PR-positive group, but this difference was only statistically significant in the T1a/b subgroup (p=0.0007). Conclusion: Further investigations into causes of p27KIP1 deregulation and their relationship to hormone receptor expression in T1N0M0 breast ductal carcinomas are warranted. Such studies may help identify prognostic, as well as predictive, markers of therapy resistance.
p27; breast cancer; node dissection; stage I; estrogen receptor; progesterone receptor
Very few reports have investigated the role of cell cycle regulators as biomarkers in Basaloid Squamous Cell Carcinoma (BSCC) of the larynx, a definite morphologic, uncommon, very aggressive variant of squamous cell carcinoma. Lower expression of Ki67/Mib-1, a proliferation marker highly expressed in the majority of tumours, and p53, a tumour suppressor protein that can induce an arrest of the G1-S transition, was related to a better prognosis in laryngeal BSCC. In the head and neck, p27kip1, a member of the Cip1/Kip1 family of cyclin-dependent kinase inhibitors, has emerged as an independent prognostic factor, able to identify low-expressing tumours with unfavourable course. Up to date the role of this protein was never studied in BSCC. Aim of our study was to investigate the potential prognostic value of p27kip1 levels and their correlation with Ki67/Mib-1 and p53 expression in BSCC of the larynx.
The retrospective study group consisted of 15 male and 1 female patients, affected by laryngeal BSCC, ranging in age from 44 to 69 years (mean 58). The tumour originated from the supraglottis in thirtheen cases and from the glottis in the remaining three. Ten patients had metastatic cervical lymph nodes at presentation and were classified as N+. Post surgical stage was IV in four patients, III in nine, II in two cases and I in the remaining one. Follow-up ranged from a minimum of 5 months up to 9 years. Paraffin-embedded tissue sections of each laryngeal tumour were analyzed for p27kip, Ki67/Mib-1 and p53 expression by immunohistochemistry.
The immunohistochemical study showed p27kip1 expression in 40% of the patients with no evidence of disease (NED) and in none (0%) of the patients dead of disease (DOD), whilst p53 was expressed in 60% of patients in NED status and in 90% of patients in DOD status. Ki67/Mib-1 was positive in 80% of NED patients and in 100% of DOD patients. At multivariate analysis, performed by means of Discriminant analysis, low levels of p27kip1 expression significantly correlated with poor prognosis (P < 0.05).
p27kip1 protein has been shown to be a significant independent prognostic factor in laryngeal SCC. In our series of laryngeal BSCC the resulting data seem to confirm the clinical prognostic relevance of p27kip1 low expression, which directly correlated with biological aggressiveness and consequent shortened survival.
The cortactin oncoprotein is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC), often due to amplification of the encoding gene (CTTN). While cortactin overexpression enhances invasive potential, recent research indicates that it also promotes cell proliferation, but how cortactin regulates the cell cycle machinery is unclear. In this article we report that stable short hairpin RNA-mediated cortactin knockdown in the 11q13-amplified cell line FaDu led to increased expression of the Cip/Kip cyclin-dependent kinase inhibitors (CDKIs) p21WAF1/Cip1, p27Kip1, and p57Kip2 and inhibition of S-phase entry. These effects were associated with increased binding of p21WAF1/Cip1 and p27Kip1 to cyclin D1- and E1-containing complexes and decreased retinoblastoma protein phosphorylation. Cortactin regulated expression of p21WAF1/Cip1 and p27Kip1 at the transcriptional and posttranscriptional levels, respectively. The direct roles of p21WAF1/Cip1, p27Kip1, and p57Kip2 downstream of cortactin were confirmed by the transient knockdown of each CDKI by specific small interfering RNAs, which led to partial rescue of cell cycle progression. Interestingly, FaDu cells with reduced cortactin levels also exhibited a significant diminution in RhoA expression and activity, together with decreased expression of Skp2, a critical component of the SCF ubiquitin ligase that targets p27Kip1 and p57Kip2 for degradation. Transient knockdown of RhoA in FaDu cells decreased expression of Skp2, enhanced the level of Cip/Kip CDKIs, and attenuated S-phase entry. These findings identify a novel mechanism for regulation of proliferation in 11q13-amplified HNSCC cells, in which overexpressed cortactin acts via RhoA to decrease expression of Cip/Kip CDKIs, and highlight Skp2 as a downstream effector for RhoA in this process.
The CDKN1B gene encodes the cyclin-dependent kinase inhibitor p27KIP1, an atypical tumor suppressor playing a key role in cell cycle regulation, cell proliferation, and differentiation. Impaired p27KIP1 expression and/or localization are often observed in tumor cells, further confirming its central role in regulating the cell cycle. Recently, germline mutations in CDKN1B have been associated with the inherited multiple endocrine neoplasia syndrome type 4, an autosomal dominant syndrome characterized by varying combinations of tumors affecting at least two endocrine organs. In this study we identified a 4-bp deletion in a highly conserved regulatory upstream ORF (uORF) in the 5′UTR of the CDKN1B gene in a patient with a pituitary adenoma and a well-differentiated pancreatic neoplasm. This deletion causes the shift of the uORF termination codon with the consequent lengthening of the uORF–encoded peptide and the drastic shortening of the intercistronic space. Our data on the immunohistochemical analysis of the patient's pancreatic lesion, functional studies based on dual-luciferase assays, site-directed mutagenesis, and on polysome profiling show a negative influence of this deletion on the translation reinitiation at the CDKN1B starting site, with a consequent reduction in p27KIP1 expression. Our findings demonstrate that, in addition to the previously described mechanisms leading to reduced p27KIP1 activity, such as degradation via the ubiquitin/proteasome pathway or non-covalent sequestration, p27KIP1 activity can also be modulated by an uORF and mutations affecting uORF could change p27KIP1 expression. This study adds the CDKN1B gene to the short list of genes for which mutations that either create, delete, or severely modify their regulatory uORFs have been associated with human diseases.
Gene expression can be modulated at different steps on the way from DNA to protein including control of transcription, translation, and post-translational modifications. An abnormality in the regulation of mRNA and protein expression is a hallmark of many human diseases, including cancer. In some eukaryotic genes translation can be influenced by small DNA sequences termed upstream open reading frames (uORFs). These elements located upstream to the gene start codon may either negatively influence the ability of the translational machinery to reinitiate translation of the main protein or, much less frequently, stimulate protein translation by enabling the ribosomes to bypass cis-acting inhibitory elements. CDKN1B, which encodes the cell cycle inhibitor p27KIP1, includes an uORF in its 5′UTR sequence. p27KIP1 expression is often reduced in cancer, and germline mutations have been identified in CDKN1B in patients affected with a syndrome (MEN4) characterized by varying combinations of tumors in endocrine glands. Here we show that a small deletion in the uORF upstream to CDKN1B reduces translation reinitiation efficiency, leading to underexpression of p27KIP1 and coinciding with tumorigenesis. This study describes a novel mechanism by which p27KIP1 could be underexpressed in human tumors. In addition, our data provide a new insight to the unique pathogenic potential of uORFs in human diseases.
HER2/neu oncogene is frequently deregulated in cancers, and the (PI3K)-Akt signaling is one of the major pathways in mediating HER2/neu oncogenic signal. p57Kip2, an inhibitor of cyclin-depependent kinases, is pivotal in regulating cell cycle progression, but its upstream regulators remain unclear. Here we show that the HER2-Akt axis is linked to p57Kip2 regulation, and that Akt is a negative regulator of p57Kip2. Ectopic expression of Akt can decrease the expression of p57Kip2, while Akt inhibition leads to p57Kip2 stabilization. Mechanistic studies show that Akt interacts with p57Kip2 and causes cytoplasmic localization of p57Kip2. Akt phosphorylates p57 on Ser 282 or Thr310. Akt activity results in destabilization of p57 by accelerating turnover rate of p57 and enhancing p57 ubiquitination. Importantly, the negative impact of HER2/Akt on p57 stability contributes to HER2-mediated cell proliferation, transformational activity and tumorigenicity. p57 restoration can attenuate these defects caused by HER2. Significantly, Kaplan-Meier analysis of tumor samples demonstrate that in tumors where HER2 expression was observed, high expression levels of p57Kip2 were associated with better overall survival. These data suggest that HER2/Akt is an important negative regulator of p57Kip2, and that p57 restoration in HER2-overexpressing cells can reduce breast tumor growth. Our findings indicate the applicability of employing p57 regulation as a therapeutic intervention in HER2-overexpressing cancers.
Akt; cell cycle; HER2; p57 Kip2; phosphorylation; subcellular localizaiton
Purpose: The cyclin-dependent kinase (Cdk) inhibitor p27Kip1 may be involved in regulating re-entry of residual hepatocytes into the cell cycle upon loss of liver tissue by partial hepatectomy (PH). As yet, changes in Kip1 expression during the initial period following PH are not well-characterized. We investigated immediate changes in Kip1 mRNA and protein levels as well as changes in Kip1 phosphorylation in liver tissue within the relevant time window between surgery and the onset of DNA synthesis at 10–12 h.
Methods: We used real-time PCR, quantitative Western blotting, and immune histochemistry on tissue samples of adult rats obtained during or between 2 and 10 h after surgical removal of two thirds of the liver to analyze Kip1 mRNA or protein levels, respectively, or to quantify nuclear expression of Kip1.
Results: Kip1 mRNA was down-regulated within 4 h after PH by 60% and remained unchanged thereafter up to 10 h. With a lag phase of 2–3 h, Kip1-protein was down-regulated to a level of 40% of the control. The level of Thr187-phosphorylated Kip1 started to increase at 4 h and reached a maximum level at 8–10 h after PH. Kip1 immunoreactivity was observed in 30% of the hepatocytes before PH. Within 6–8 h after PH, more than half of the hepatocytes lost nuclear Kip1 signals. Kip1-specific micro-RNAs (miRNA221, miRNA222) were not changed upon PH.
Conclusions: A portion of hepatocytes in adult rats constitutively express Kip1 and down-regulate Kip1 immediately upon PH. This response involves transcriptional processes (loss of Kip1 mRNA) as well as accelerated degradation of existing protein (increase in pThr187-phosphorylation mediating polyubiquitinylation and proteasomal degradation of Kip1). Kip1 down-regulation occurs precisely within the intervall between surgery and onset of DNA synthesis which supports the hypothesis that it mediates activation of G0/0S-phase Cdk/cyclin-complexes and re-entry of hepatocytes into the cell cycle.
cell cycle regulator; cyclin-dependent kinase inhibitor; Kip1; compensatory growth; liver regeneration; rat hepatocytes; cell proliferation
Eukaryotic initiation factor 4E (eIF4E) is essential for cap-dependent initiation of translation. Cell proliferation is associated with increased activity of eIF4E and elevated expression of eIF4E leads to tumorigenic transformation. Many tumors express very high levels of eIF4E and this may be a critical factor in progression of the disease. In contrast, overexpression of 4EBP, an inhibitor of eIF4E, leads to cell cycle arrest and phenotypic reversion of some transformed cells.
A constitutively active form of 4EBP-1 was inducibly expressed in the human breast cancer cell line MCF7. Induction of constitutively active 4EBP-1 led to cell cycle arrest. This was not associated with a general inhibition of protein synthesis but rather with changes in specific cell cycle regulatory proteins. Cyclin D1 was downregulated while levels of the CDK inhibitor p27Kip1 were increased. The levels of cyclin E and CDK2 were unaffected but the activity of CDK2 was significantly reduced due to increased association with p27Kip1. The increase in p27Kip1 did not reflect changes in p27Kip1 mRNA or degradation rates. Rather, it was associated with enhanced synthesis of the protein, even though 4EBP-1 is expected to inhibit translation. This could be explained, at least in part, by the ability of the p27Kip1 5'-UTR to mediate cap-independent translation, which was also enhanced by expression of constitutively active 4EBP-1.
Expression of active 4EBP-1 in MCF7 leads to cell cycle arrest which is associated with downregulation of cyclin D1 and upregulation of p27Kip1. Upregulation of p27Kip1reflects increased synthesis which corresponds to enhanced cap-independent translation through the 5'-UTR of the p27Kip1 mRNA.
Neuroblastoma (NB) is the most common extracranial pediatric tumor. NB patients over 18 months of age at the time of diagnosis are often in the later stages of the disease, present with widespread dissemination, and often possess MYCN tumor gene amplification. MYCN is a transcription factor that regulates the expression of a number of genes including ornithine decarboxylase (ODC), a rate-limiting enzyme in the biosynthesis of polyamines. Inhibiting ODC in NB cells produces many deleterious effects including G1 cell cycle arrest, inhibition of cell proliferation, and decreased tumor growth, making ODC a promising target for drug interference. DFMO treatment leads to the accumulation of the cyclin-dependent kinase inhibitor p27Kip1 protein and causes p27Kip1/Rb-coupled G1 cell cycle arrest in MYCN-amplified NB tumor cells through a process that involves p27Kip1 phosphorylation at residues Ser10 and Thr198. While p27Kip1 is well known for its role as a cyclin-dependent kinase inhibitor, recent studies have revealed a novel function of p27Kip1 as a regulator of cell migration and invasion. In the present study we found that p27Kip1 regulates the migration and invasion in NB and that these events are dependent on the state of phosphorylation of p27Kip1. DFMO treatments induced MYCN protein downregulation and phosphorylation of Akt/PKB (Ser473) and GSK3-β (Ser9), and polyamine supplementation alleviated the DFMO-induced effects. Importantly, we provide strong evidence that p27Kip1 mRNA correlates with clinical features and the survival probability of NB patients.
DFMO; Kaplan-Meier survival plot; metastasis; MYCN; neuroblastoma; ornithine decarboxylase; polyamines; p27Kip
The universal cyclin-Cdk inhibitor p27Kip1 functions as a tumor suppressor and reduced levels of p27Kip1 connote poor prognosis in several human malignancies. p27Kip1 levels are predominately regulated by ubiquitin-mediated turnover of the protein, which is marked for destruction by the E3 ubiquitin ligase SCFSkp2 complex following its phosphorylation by the cyclin E-Cdk2 complex. Binding of phospho-p27Kip1 is directed by the Skp2 F-box protein, and this is greatly augmented by its allosteric regulator Cks1. We have established that programmed expression of c-Myc in the B cells of Eμ-Myc transgenic mice triggers p27Kip1 destruction by inducing Cks1, that this response controls Myc-driven proliferation, and that loss of Cks1 markedly delays Myc-induced lymphomagenesis and cancels the dissemination of these tumors. Here, we report that elevated levels of Skp2 are a characteristic of Eμ-Myc lymphomas and of human Burkitt lymphoma that bear MYC/immunoglobulin chromosomal translocations. As expected, Myc-mediated suppression of p27Kip1 was abolished in Skp2-null Eμ-Myc B cells. However, the impact of Skp2 loss on Myc-driven proliferation and lymphomagenesis was surprisingly modest compared to the effects of Cks1 loss. Collectively these findings suggest that Cks1 targets in addition to p27Kip1 are critical for Myc-driven proliferation and tumorigenesis.
Myc; Skp2; p27Kip1; lymphomagenesis
Hypoxia induces the proliferation of pulmonary arterial smooth muscle cell (PASMC) in vivo and in vitro, and prostacyclin analogues are thought to inhibit the growth of PASMC. Previous studies suggest that p27kip1, a kind of cyclin-dependent kinase inhibitor, play an important role in the smooth muscle cell proliferation. However, the mechanism of hypoxia and the subcellular interactions between p27kip1 and prostacyclin analogues in human pulmonary arterial smooth muscle cell (HPASMC) are not fully understood.
We investigated the role of p27kip1 in the ability of Beraprost sodium (BPS; a stable prostacyclin analogue) to inhibit the proliferation of HPASMC during hypoxia. To clarify the biological effects of hypoxic air exposure and BPS on HPASMC, the cells were cultured in a hypoxic chamber under various oxygen concentrations (0.1–21%). Thereafter, DNA synthesis was measured as bromodeoxyuridine (BrdU) incorporation, the cell cycle was analyzed by flow cytometry with propidium iodide staining. The p27kip1 mRNA and protein expression and it's stability was measured by real-time RT-PCR and Western blotting. Further, we assessed the role of p27kip1 in HPASMC proliferation using p27kip1 gene knockdown using small interfering RNA (siRNA) transfection.
Although severe hypoxia (0.1% oxygen) suppressed the proliferation of serum-stimulated HPASMC, moderate hypoxia (2% oxygen) enhanced proliferation in accordance with enhanced p27kip1 protein degradation, whereas BPS suppressed HPASMC proliferation under both hypoxic and normoxic conditions by suppressing p27kip1 degradation with intracellular cAMP-elevation. The 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), a cAMP analogue, had similar action as BPS in the regulation of p27kip1. Moderate hypoxia did not affect the stability of p27kip1 protein expression, but PDGF, known as major hypoxia-induced growth factors, significantly decreased p27kip1 protein stability. We also demonstrated that BPS and 8-Br-cAMP suppressed HPASMC proliferation under both hypoxic and normoxic conditions by blocking p27kip1 mRNA degradation. Furthermore, p27kip1 gene silencing partially attenuated the effects of BPS and partially restored hypoxia-induced proliferation.
Our study suggests that moderate hypoxia induces HPASMC proliferation, which is partially dependent of p27kip1 down-regulation probably via the induction of growth factors such as PDGF, and BPS inhibits both the cell proliferation and p27kip1 mRNA degradation through cAMP pathway.
In developing central nervous system, a variety of mechanisms couple cell cycle exit to differentiation during neurogenesis. The cyclin-dependent kinase (CDK) inhibitor p57Kip2 controls the transition from proliferation to differentiation in many tissues, but roles in developing brain remain uncertain. To characterize possible functions, we defined p57Kip2 protein expression in embryonic day (E) 12.5 to 20.5 rat brains using immunohistochemistry combined with markers of proliferation and differentiation. p57Kip2 was localized primarily in cell nuclei and positive cells formed two distinct patterns including wide dispersion and laminar aggregation that were brain region-specific. From E12.5 to E16.5, p57Kip2 expression was detected mainly in ventricular (VZ) and/or mantle zones of hippocampus, septum, basal ganglia, thalamus, hypothalamus, midbrain and spinal cord. After E18.5, p57Kip2 was detected in select regions undergoing differentiation. p57Kip2 expression was also compared to regional transcription factors, including Ngn2, Nkx2.1 and Pax6. Time course studies performed in diencephalon showed that p57Kip2 immunoreactivity co-localized with BrdU at 8 hr in nuclei exhibiting the wide dispersion pattern, whereas co-localization in the laminar pattern occurred only later. Moreover, p57Kip2 frequently co-localized with neuronal marker, β-III tubulin. Finally, we characterized relationships of p57Kip2 to CDK inhibitor p27Kip1: In proliferative regions, p57Kip2 expression preceded p27Kip1 as cells underwent differentiation, though the proteins co-localized in substantial numbers of cells, suggesting potentially related yet distinct functions of Cip/Kip family members during neurogenesis. Our observations that p57Kip2 exhibits nuclear expression as precursors exit the cell cycle and begin expressing neuronal characteristics suggests that the CDK inhibitor contributes to regulating the transition from proliferation to differentiation during brain development.
Cyclin-Dependent Kinase Inhibitor p57Kip2; Embryonic Development/physiology; Nervous System/cytology/*embryology; Brain/embryology; Neuronal Differentiation
p27Kip1 is a cell cycle inhibitor that prevents cyclin dependent kinase (CDK)/cyclin complexes from phosphorylating their targets. p27Kip1 is a known tumor suppressor, as the germline loss of p27Kip1 results in sporadic pituitary formation in aged rodents, and its presence in human cancers is indicative of a poor prognosis. In addition to its role in cancer, loss of p27Kip1 results in regenerative phenotypes in some tissues and maintenance of stem cell pluripotency, suggesting that p27Kip1 inhibitors could be beneficial for tissue regeneration. Because p27Kip1 is an intrinsically disordered protein, identifying direct inhibitors of the p27Kip1 protein is difficult. Therefore, we pursued a high-throughput screening strategy to identify novel p27Kip1 transcriptional inhibitors. We utilized a luciferase reporter plasmid driven by the p27Kip1 promoter to transiently transfect HeLa cells and used cyclohexamide as a positive control for non-specific inhibition. We screened a “bioactive” library consisting of 8,904 (4,359 unique) compounds, of which 830 are Food and Drug Administration (FDA) approved. From this screen, we successfully identified 111 primary hits with inhibitory effect against the promoter of p27Kip1. These hits were further refined using a battery of secondary screens. Here we report four novel p27Kip1 transcriptional inhibitors, and further demonstrate that our most potent hit compound (IC50 = 200 nM) Alsterpaullone 2-cyanoethyl, inhibits p27Kip1 transcription by preventing FoxO3a from binding to the p27Kip1 promoter. This screen represents one of the first attempts to identify inhibitors of p27Kip1 and may prove useful for future tissue regeneration studies.
The cyclin/cyclin-dependent kinase (cdk) inhibitor p27kip1 is thought to be responsible for the onset and maintenance of the quiescent state. It is possible, however, that cells respond differently to p27kip1 in different conditions, and using a BALB/c-3T3 cell line (termed p27-47) that inducibly expresses high levels of this protein, we show that the effect of p27kip1 on cell cycle traverse is determined by cell density. We found that ectopic expression of p27kip1 blocked the proliferation of p27-47 cells at high density but had little effect on the growth of cells at low density whether exponentially cycling or stimulated from quiescence. Regardless of cell density, the activities of cdk4 and cdk2 were markedly repressed by p27kip1 expression, as was the cdk4-dependent dissociation of E2F4/p130 complexes. Infection of cells with SV40, a DNA tumor virus known to abrogate formation of p130- and Rb-containing complexes, allowed dense cultures to proliferate in the presence of supraphysiological amounts of p27kip1 but did not stimulate cell cycle traverse when cultures were cotreated with the potent cdk2 inhibitor roscovitine. Our data suggest that residual levels of cyclin/cdk activity persist in p27kip1-expressing p27-47 cells and are sufficient for the growth of low-density cells and of high-density cells infected with SV40, and that effective disruption of p130 and/or Rb complexes is obligatory for the proliferation of high-density cultures.
We describe the design of a potent and selective peptidomimetic inhibitor of geranylgeranyltransferase I (GGTI), GGTI-2418, and its methyl ester GGTI-2417, which increases the levels of the cyclin-dependent kinase (Cdk) inhibitor p27Kip1 and induces breast tumor regression in vivo. Experiments with p27Kip1 small interfering RNA in breast cancer cells and p27Kip1 null murine embryonic fibroblasts demonstrate that the ability of GGTI-2417 to induce cell death requires p27Kip1. GGTI-2417 inhibits the Cdk2-mediated phosphorylation of p27Kip1 at Thr187 and accumulates p27Kip1 in the nucleus. In nude mouse xenografts, GGTI-2418 suppresses the growth of human breast tumors. Furthermore, in ErbB2 transgenic mice, GGTI-2418 increases p27Kip1 and induces significant regression of breast tumors. We conclude that GGTIs' antitumor activity is, at least in part, due to inhibiting Cdk2-dependent p27Kip1 phosphorylation at Thr187 and accumulating nuclear p27Kip1. Thus, GGTI treatment might improve the poor prognosis of breast cancer patients with low nuclear p27Kip1 levels.