P27 Kip1 Promoter Research Articles

Decreased proliferation of aged rat beta cells corresponds with enhanced expression of the cell cycle inhibitor p27KIP1.

Over 400 million people are diabetic. Type 1 and type 2 diabetes are characterized by decreased functional β-cell mass and, consequently, decreased glucose-stimulated insulin secretion. A potential intervention is transplantation of β-cell containing islets from cadaveric donors. A major impediment to greater application of this treatment is the scarcity of transplant-ready β-cells. Therefore, inducing β-cell proliferation ex vivo could be used to expand functional β-cell mass prior to transplantation. Various molecular pathways are sufficient to induce proliferation of young β-cells; however, aged β-cells are refractory to these proliferative signals. Given that the majority of cadaveric donors fit an aged demographic, defining the mechanisms that impede aged β-cell proliferation is imperative. We demonstrate that aged rat (5-month-old) β-cells are refractory to mitogenic stimuli that otherwise induce young rat (5-week-old) β-cell proliferation. We hypothesized that this change in proliferative capacity could be due to differences in cyclin-dependent kinase inhibitor expression. We measured levels of p16INK4a , p15INK4b , p18INK4c , p19INK4d , p21CIP1 , p27KIP1 and p57KIP2 by immunofluorescence analysis. Our data demonstrates an age-dependent increase of p27KIP1 in rat β-cells by immunofluorescence and was validated by increased p27KIP1 protein levels by western blot analysis. Interestingly, HDAC1, which modulates the p27KIP1 promoter acetylation state, is downregulated in aged rat islets. These data demonstrate increased p27KIP1 protein levels at 5 months of age, which may be due to decreased HDAC1 mediated repression of p27KIP1 expression. As the majority of transplant-ready β-cells come from aged donors, it is imperative that we understand why aged β-cells are refractory to mitogenic stimuli. Our findings demonstrate that increased p27KIP1 expression occurs early in β-cell aging, which corresponds with impaired β-cell proliferation. Furthermore, the correlation between HDAC1 and p27 levels suggests that pathways that activate HDAC1 in aged β-cells could be leveraged to decrease p27KIP1 levels and enhance β-cell proliferation.

GATA3 maintains the quiescent state of cochlear supporting cells by regulating p27kip1

Haplo-insufficiency of the GATA3 gene causes hypoparathyroidism, sensorineural hearing loss, and renal disease (HDR) syndrome. Previous studies have shown that Gata3 is required for the development of the prosensory domain and spiral ganglion neurons (SGNs) of the mouse cochlea during embryogenesis. However, its role in supporting cells (SCs) after cell fate specification is largely unknown. In this study, we used tamoxifen-inducible Sox2CreERT2 mice to delete Gata3 in SCs of the neonatal mouse cochlea and showed that loss of Gata3 resulted in the proliferation of SCs, including the inner pillar cells (IPCs), inner border cells (IBCs), and lateral greater epithelium ridge (GER). In addition, loss of Gata3 resulted in the down-regulation of p27kip1, a cell cycle inhibitor, in the SCs of Gata3-CKO neonatal cochleae. Chromatin immunoprecipitation analysis revealed that GATA3 directly binds to p27kip1 promoter and could maintain the quiescent state of cochlear SCs by regulating p27kip1 expression. Furthermore, RNA-seq analysis revealed that loss of Gata3 function resulted in the change in the expression of genes essential for the development and function of cochlear SCs, including Tectb, Cyp26b1, Slitrk6, Ano1, and Aqp4.

Silencing heme oxygenase-1 increases the sensitivity of ABC-DLBCL cells to histone deacetylase inhibitor in vitro and in vivo

Heme oxygenase-1 (HO-1) can promote tumor growth and reinforce the resistance of diffuse large B-cell lymphoma (DLBCL) cells to chemotherapeutic drug vincristine. We herein found that HO-1 protein expression was higher in high-risk DLBCL patients than in low-risk ones. Silencing HO-1 gene expression resisted vorinostat-induced apoptosis and arrested cell cycle in the G0/G1 phase of LY-10 cells. Western blot, co-immunoprecipitation and chromatin immunoprecipitation assays confirmed that the possible mechanisms may be increased cleaved caspase-3 protein expression, decreased phospho-histone deacetylase 3 protein expression, and activated histone acetylation of P27Kip1 promoter. Moreover, silencing HO-1 gene expression enhanced vorinostat-induced tumor cell apoptosis, prolonged survival time and promoted P27Kip1 protein expression in a xenograft mouse model.In conclusion, HO-1 is a potential therapeutic target of DLBCL. The findings provide a valuable preclinical evidence for sensitizing DLBCL patients with poor prognosis to histone deacetylase inhibitors.

Synergistic effect of melatonin and ghrelin in preventing cisplatin-induced ovarian damage via regulation of FOXO3a phosphorylation and binding to the p27Kip1 promoter in primordial follicles.

Premature ovarian failure during chemotherapy is a serious problem for young women with cancer. To preserve the fertility of these patients, approaches to prevent chemotherapy-induced ovarian failure are needed. In a previous study, we reported that melatonin treatment prevents the depletion of the dormant follicle pool via repression of the simultaneous activation of dormant primordial follicles by cisplatin. However, melatonin's protective effect was only partial and thus insufficient. In this study, we found that the hormone ghrelin enhances the protective effect of melatonin against cisplatin-induced ovarian failure in mouse model. Co-administration of melatonin and ghrelin more effectively prevented cisplatin-induced follicle disruption. Simultaneous treatment with melatonin and ghrelin almost restored the number of primordial follicles and the corpus luteum in cisplatin-treated ovaries, compared with single administration. We found melatonin and ghrelin receptors on the cell membrane of premature oocytes of primordial follicles. In addition, melatonin and ghrelin co-administration inhibited the cisplatin-induced phosphorylation of PTEN and FOXO3a that induces cytoplasmic translocation of FOXO3a. Inhibition of FOXO3a phosphorylation by melatonin and ghrelin increased the binding affinity of FOXO3a for the p27Kip1 promoter in primordial follicles. Co-administration of melatonin and ghrelin in cisplatin-treated ovaries restored the expression of p27Kip1 , which is critical for retention of the dormant status of primordial follicles. In conclusion, these findings suggest that melatonin and ghrelin co-administration is suitable for use as a fertoprotective adjuvant therapy during cisplatin chemotherapy in young female cancer patients.

LncRNA GAS5 Inhibits Cellular Proliferation by Targeting P27Kip1

Recent studies have demonstrated that long noncoding RNAs (lncRNA) have important roles in cancer biology, and that the downregulation of lncRNA growth arrest-specific transcript 5 (GAS5) has been reported in a variety of human cancers. However, its role in prostate cancer is largely unknown. This study aims to investigate the biological role and underlying mechanism of GAS5 on proliferation in prostate cancer. The results demonstrate that GAS5 expression is significantly decreased in prostate cancer cells compared with prostate epithelial cells. Ectopic expression of GAS5 inhibited cell proliferation and induced a cell-cycle arrest in G0-G1 phase, whereas GAS5 knockdown promoted the G1-S phase transition. Subsequent analysis demonstrated that P27Kip1, a known regulator of cell cycle, was positively regulated by GAS5 and upregulation of GAS5 increased its promoter activity. E2F1, an important transcription factor, was shown to bind directly to and activate the P27Kip1 promoter. In addition, GAS5 interacted with E2F1 and enhanced the binding of E2F1 to the P27Kip1 promoter. Collectively, these findings determine that GAS5 functions as a tumor suppressor in prostate cancer development and progression via targeting P27Kip1Implications: This study reveals a molecular pathway involving lncRNA GAS5/E2F1/P27Kip1 which regulates cell proliferation and could be a potential therapeutic target in prostate cancer. Mol Cancer Res; 15(7); 789-99. ©2017 AACR.

The functional variant rs34330 of CDKN1B is associated with risk of neuroblastoma.

The genetic aetiology of sporadic neuroblastoma is still largely unknown. We have identified diverse neuroblastoma susceptibility loci by genomewide association studies (GWASs); however, additional SNPs that likely contribute to neuroblastoma susceptibility prompted this investigation for identification of additional variants that are likely hidden among signals discarded by the multiple testing corrections used in the analysis of genomewide data. There is evidence suggesting the CDKN1B, coding for the cycle inhibitor p27Kip1, is involved in neuroblastoma. We thus assess whether genetic variants of CDKN1B are associated with neuroblastoma. We imputed all possible genotypes across CDKN1B locus on a discovery case series of 2101 neuroblastoma patients and 4202 genetically matched controls of European ancestry. The most significantly associated rs34330 was analysed in an independent Italian cohort of 311 cases and 709 controls. In vitro functional analysis was carried out in HEK293T and in neuroblastoma cell line SHEP‐2, both transfected with pGL3‐CDKN1B‐CC or pGL3‐CDKN1B‐TT constructs. We identified an association of the rs34330 T allele (‐79C/T) with the neuroblastoma risk (Pcombined = 0.002; OR = 1.17). The risk allele (T) of this single nucleotide polymorphism led to a lower transcription rate in cells transfected with a luciferase reporter driven by the polymorphic p27Kip1 promoter (P < 0.05). Three independent sets of neuroblastoma tumours carrying ‐79TT genotype showed a tendency towards lower CDKN1B mRNA levels. Our study shows that a functional variant, associated with a reduced CDKN1B gene transcription, influences neuroblastoma susceptibility.

HBx-upregulated lncRNA UCA1 promotes cell growth and tumorigenesis by recruiting EZH2 and repressing p27Kip1/CDK2 signaling.

It is well accepted that HBx plays the major role in hepatocarcinogenesis associated with hepatitis B virus (HBV) infections. However, little was known about its role in regulating long noncoding RNAs (lncRNAs), a large group of transcripts regulating a variety of biological processes including carcinogenesis in mammalian cells. Here we report that HBx upregulates UCA1 genes and downregulates p27 genes in hepatic LO2 cells. Further studies show that the upregulated UCA1 promotes cell growth by facilitating G1/S transition through CDK2 in both hepatic and hepatoma cells. Knock down of UCA1 in HBx-expressing hepatic and hepatoma cells resulted in markedly increased apoptotic cells by elevating the cleaved caspase-3 and caspase-8. More importantly, UCA1 is found to be physically associated with enhancer of zeste homolog 2 (EZH2), which suppresses p27Kip1 through histone methylation (H3K27me3) on p27Kip1 promoter. We also show that knockdown of UCA1 in hepatoma cells inhibits tumorigenesis in nude mice. In a clinic study, UCA1 is found to be frequently up-regulated in HBx positive group tissues in comparison with the HBx negative group, and exhibits an inverse correlation between UCA1 and p27Kip1 levels. Our findings demonstrate an important mechanism of hepatocarcinogenesis through the signaling of HBx-UCA1/EZH2-p27Kip1 axis, and a potential target of HCC.

Abstract 165: The Transcriptional Cofactor Eyes Absent 4 Is A Critical Regulator In Cardiac Physiology

Introduction: Eyes absent 4 (Eya4) is a transcription cofactor involved in a number of cellular and developmental processes. We have previously shown that a truncating mutation in Eya4, E193, causes hearing impairment followed by terminal heart failure suggesting Eya4 is a regulator in cardiac physiology. Methods and Results: Transgene- or adenovirus mediated overexpression of Eya4 or E193 altered the expression of p27kip1, a critical mediator of cardiac hypertrophy in adult cardiomyocytes. Luciferase reporter and EMSA assays revealed that Eya4 directly binds to and suppresses p27kip1 promoter activity, while E193 exerts an opposing effect, respectively. Activity and phosphorylation of downstream molecules were significantly altered in Eya4 and E193 transgenic (TG) mice in a contradictory manner. Cardiac phenotypes evolved in both TG models already under basal conditions. Eya4 TG hearts developed hypertrophy as judged by increases in heart weight and cross-sectional cell surfaces and re-activation of fetal genes as well as fibrosis. E193 TG animals showed onset of DCM along with wall thinning, ventricular dilation, fibrosis and slightly compromised cardiac function. These two distinct cardiac phenotypes were even more aggravated upon pressure overload or Angiotensin II infusion. Finally, we just recently identified a new mutation in Eya4, E215, which also causes hearing impairment and heart failure. Conclusion: Our data indicate that Eya4, in a physical complex with Six1, plays a critical role in regulating normal cardiac function via p27/CK2-α/HDAC2 and allude that mutations within the Exa4/Six1 transcriptional complex interfere with this newly established signalling pathway, finally leading to age-related onset of cardiomyopathy. Clinical Perspective: Gaining a better understanding of this disease mechanism could help identify new treatment options for heart failure patients.

High-Throughput Screening Reveals Alsterpaullone, 2-Cyanoethyl as a Potent p27Kip1 Transcriptional Inhibitor

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.

Promoter methylation-associated silencing of p27kip1 gene with metastasis in esophageal squamous cell carcinoma

The aim of the present study was to determine the frequency of p27kip1 promoter methylation in esophageal squamous cell carcinoma (ESCC). The methylation status of the p27kip1 promoter was analyzed by methylation‑specific polymerase chain reaction (MSP) in 50 ESCC and matched non‑tumor tissues. Cell lines were treated with the demethylation agent 5‑aza‑2'‑deoxycytidine (5‑Aza‑CdR) and p27kip1 mRNA expression was detected by quantitative polymerase chain reaction. p27kip1 methylation was found in 36% (18/50) of ESCC patients, but only in 12% (6/50) of the corresponding non‑tumor tissues (P=0.005). There were statistically significant associations between the presence of methylation and tumor metastasis (P=0.002). The p27kip1 mRNA was lower in ESCC compared with non‑tumor tissues (mean ± standard deviation, ‑0.886±3.298 vs. 0.988±0.257; P=0.0033). Furthermore, a significant association was identified between the methylation status of the p27kip1 promoter and p27kip1 mRNA expression in the tissue (P<0.01). Thus, demethylation by 5‑Aza‑CdR was capable of inducing p27kip1 mRNA expression in esophageal cancer cell lines. The high promoter methylation of p27kip1 is a common phenomenon in ESCC, which may be an important mechanism of silencing p27kip1 mRNA expression.

Undifferentiated embryonic cell transcription factor-1 (UTF1) inhibits the growth of cervical cancer cells by transactivating p27Kip1

Undifferentiated embryonic cell transcription factor-1 (UTF1) is an important transcription factor during development, which plays critical roles in cell fate determination. However, its expression and function in somatic tissues remain unclear. Here, we investigated the expression pattern of the UTF1 in the human normal and cancerous lesions of cervix and found that UTF1 was downregulated in cervical carcinogenesis, which was related to the hypermethylation of UTF1 promoter. Exogenous expression of UTF1 resulted in the significant inhibition of cell proliferation in vitro and tumorigenesis in vivo through attenuating cell cycle arrest via increasing the level of p27 (Kip1) . Luciferase reporter assay indicated that the region containing an intact activating transcription factor site between nucleotides -517 and -388 of the p27 (Kip1) promoter was indispensable for its activation by UTF1. Chromatin immunoprecipitation analysis confirmed the physical interaction between UTF1 and the p27 (Kip1) promoter. Taken together, our findings reveal that UTF1 attenuates cell proliferation and is inactivated in cervical carcinogenesis through epigenetic modification, which strongly supports that UTF1 is a potential tumor suppressor.

Peroxisome Proliferator-activated Receptor γ Agonists Induce Cell Cycle Arrest through Transcriptional Regulation of Krüppel-like Factor 4 (KLF4)

Peroxisome proliferator-activated receptor γ (PPARγ), a subgroup of ligand-activated nuclear receptors, plays critical roles in cell cycle regulation, differentiation, apoptosis, and invasion. PPARγ is involved in tumorigenesis and is a potent target for cancer therapy. PPARγ transactivation of KLF4 has been demonstrated in various studies; however, how PPARγ regulates KLF4 expression is not clear. In this study, we reveal that PPARγ regulates the expression of KLF4 by binding directly to the PPAR response element (PPRE) within the KLF4 promoter. The PPRE resides at -1657 to -1669 bp upstream of the KLF4 ATG codon, which is essential for the transactivation of troglitazone-induced KLF4 expression. Furthermore, we found that stable silencing of KLF4 obviously suppressed the G(1)/S arrest and anti-proliferation effects induced by PPARγ ligands. Taken together, our data indicate that up-regulation of KLF4 upon PPARγ activation is mediated through the PPRE in the KLF4 promoter, thus providing further insights into the PPARγ signal transduction pathway as well as a novel cancer therapeutic strategy.

Melatonin MT1 receptor‐induced transcriptional up‐regulation of p27Kip1 in prostate cancer antiproliferation is mediated via inhibition of constitutively active nuclear factor kappa B (NF‐κB): potential implications on prostate cancer chemoprevention and therapy

Our laboratory has recently demonstrated a melatonin MT1 receptor-mediated antiproliferative signaling mechanism in androgen receptor (AR)-positive prostate epithelial cells which involves up-regulation of p27(Kip1) through dual activation of Gα(s)/protein kinase A (PKA) and Gα(q)/protein kinase C (PKC) in parallel, and down-regulation of activated AR signaling via PKC stimulation. The aim of the present investigation was to identify the transcription factor that mediates melatonin's up-regulatory effect on p27(Kip1) in LNCaP and 22Rv1 prostate cancer cells. Deletion mapping and reporter assays of the p27(Kip1) promoter revealed that the putative melatonin-responsive transcription factor binds to a 116 base-pair region of the promoter sequence, which contains a potential nuclear factor kappa B (NF-κB) binding site. When the NF-κB binding site was abolished by site-directed mutagenesis, the stimulatory effect of melatonin on p27(Kip1) promoter activity was mitigated. Notably, melatonin inhibited the DNA binding of activated NF-κB via MT1 receptor-induced PKA and PKC stimulation. Furthermore, melatonin's up-regulatory effect on p27(Kip1) transcription and consequent cell antiproliferation were abrogated by NF-κB activator but mimicked by NF-κB inhibitor. The results indicate that inhibition of constitutively active NF-κB via melatonin MT1 receptor-induced dual activation of (Gα(s)) PKA and (Gα(q)) PKC can de-repress the p27(Kip1) promoter leading to transcriptional up-regulation of p27(Kip1). MT1 receptor-mediated inhibition of activated NF-κB signaling provides a novel mechanism supporting the use of melatonin in prostate cancer chemoprevention and therapy.

Regulation of p27Kip1 by Sox2 Maintains Quiescence of Inner Pillar Cells in the Murine Auditory Sensory Epithelium

Sox2 plays critical roles in cell fate specification during development and in stem cell formation; however, its role in postmitotic cells is largely unknown. Sox2 is highly expressed in supporting cells (SCs) of the postnatal mammalian auditory sensory epithelium, which unlike non-mammalian vertebrates remains quiescent even after sensory hair cell damage. Here, we induced the ablation of Sox2, specifically in SCs at three different postnatal ages (neonatal, juvenile and adult) in mice. In neonatal mice, Sox2-null inner pillar cells (IPCs, a subtype of SCs) proliferated and generated daughter cells, while other SC subtypes remained quiescent. Furthermore, p27(Kip1), a cell cycle inhibitor, was absent in Sox2-null IPCs. Similarly, upon direct deletion of p27(Kip1), p27(Kip1)-null IPCs also proliferated but retained Sox2 expression. Interestingly, cell cycle control of IPCs by Sox2-mediated expression of p27(Kip1) gradually declined with age. In addition, deletion of Sox2 or p27(Kip1) did not cause a cell fate change. Finally, chromatin immunoprecipitation with Sox2 antibodies and luciferase reporter assays with the p27(Kip1) promoter support that Sox2 directly activates p27(Kip1) transcription in postmitotic IPCs. Hence, in contrast to the well known activity of Sox2 in promoting proliferation and cell fate determination, our data demonstrate that Sox2 plays a novel role as a key upstream regulator of p27(Kip1) to maintain the quiescent state of postmitotic IPCs. Our studies suggest that manipulating Sox2 or p27(Kip1) expression is an effective approach to inducing proliferation of neonatal auditory IPCs, an initial but necessary step toward restoring hearing in mammals.

Krüppel‐like factor 4 functions as a tumor suppressor in cervical carcinoma

The Krüppel-like factor 4 (KLF4) protein, a zinc finger transcription factor that is highly expressed in epithelial tissues such as the gut and skin, has been implicated in both tumor suppression and progression. However, the role of KLF4 in human cervical carcinoma is still unclear. The expression of KLF4 in cervical carcinoma tissues and cervical cancer cell lines was examined with immunohistochemistry and Western blot assay. The effects of KLF4 silencing and overexpression on the cell proliferation, cell viability, and tumor formation of cervical cancer cells were investigated. KLF4 protein expression showed a pattern of gradual decrease from normal cervix to cervical carcinoma in situ and then to invasive cervical carcinomas (P < .05). Overexpression of KLF4 in SiHa and C33A cells resulted in significantly inhibited cell growth and significantly attenuated tumor formation. Consistently, KLF4 silencing in HeLa cells significantly promoted cell growth and tumor formation. Furthermore, KLF4 overexpression caused cell cycle arrest at the G1/S transition, along with the up-regulated expression of p27(Kip1) protein. Promoter analysis revealed that KLF4 transactivated the expression of p27(Kip1) through the specific motif that is between the nucleotides of -435 and -60 in its promoter. The results from chromatin immunoprecipitation assays demonstrated the physical interaction between KLF4 protein and this specific motif in p27(Kip1) promoter. KLF4 may function as a tumor suppressor in cervical carcinoma by inhibiting cell growth and tumor formation.

Genistein inhibits proliferation of colon cancer cells by attenuating a negative effect of epidermal growth factor on tumor suppressor FOXO3 activity

BackgroundSoy consumption is associated with a lower incidence of colon cancer which is believed to be mediated by one of its of components, genistein. Genistein may inhibit cancer progression by inducing apoptosis or inhibiting proliferation, but mechanisms are not well understood. Epidermal growth factor (EGF)-induced proliferation of colon cancer cells plays an important role in colon cancer progression and is mediated by loss of tumor suppressor FOXO3 activity. The aim of this study was to assess if genistein exerts anti-proliferative properties by attenuating the negative effect of EGF on FOXO3 activity.MethodsThe effect of genistein on proliferation stimulated by EGF-mediated loss of FOXO3 was examined in human colonic cancer HT-29 cells. EGF-induced FOXO3 phosphorylation and translocation were assessed in the presence of genistein. EGF-mediated loss of FOXO3 interactions with p53 (co-immunoprecipitation) and promoter of p27kip1 (ChIP assay) were examined in presence of genistein in cells with mutated p53 (HT-29) and wild type p53 (HCT116). Silencing of p53 determined activity of FOXO3 when it is bound to p53.ResultsGenistein inhibited EGF-induced proliferation, while favoring dephosphorylation and nuclear retention of FOXO3 (active state) in colon cancer cells. Upstream of FOXO3, genistein acts via the PI3K/Akt pathway to inhibit EGF-stimulated FOXO3 phosphorylation (i.e. favors active state). Downstream, EGF-induced disassociation of FOXO3 from mutated tumor suppressor p53, but not wild type p53, is inhibited by genistein favoring FOXO3-p53(mut) interactions with the promoter of the cell cycle inhibitor p27kip1 in colon cancer cells. Thus, the FOXO3-p53(mut) complex leads to elevated p27kip1 expression and promotes cell cycle arrest.ConclusionThese novel anti-proliferative mechanisms of genistein suggest a possible role of combining genistein with other chemoreceptive agents for the treatment of colon cancer.

The cell cycle inhibitor p27Kip1 controls self-renewal and pluripotency of human embryonic stem cells by regulating the cell cycle, Brachyury and Twist

The continued turn over of human embryonic stem cells (hESC) while maintaining an undifferentiated state is dependent on the regulation of the cell cycle. Here we asked the question if a single cell cycle gene could regulate the self-renewal or pluripotency properties of hESC. We identified that the protein expression of the p27Kip1 cell cycle inhibitor is low in hESC cells and increased with differentiation. By adopting a gain and loss of function strategy we forced or reduced its expression in undifferentiating conditions to define its functional role in self-renewal and pluripotency. Using undifferentiation conditions, overexpression of p27Kip1 in hESC lead to a G1 phase arrest with an enlarged and flattened hESC morphology and consequent loss of self-renewal ability. Loss of p27Kip1 caused an elongated/scatter cell-like phenotype involving upregulation of Brachyury and Twist gene expression. We demonstrate the novel finding that p27Kip1 protein occupies the Twist1 gene promoter and manipulation of p27Kip1 by gain and loss of function is associated with Twist gene expression changes. These results define p27Kip1 expression levels as critical for self-renewal and pluripotency in hESC and suggest a role for p27Kip1 in controlling an epithelial to mesenchymal transition (EMT) in hESC.

Tumor Suppressor Menin Represses Paired Box Gene 2 Expression via Wilms Tumor Suppressor Protein-Polycomb Group Complex

Tumor suppressor menin, the product of the MEN1 gene, plays a key role in controlling histone 3 lysine 4 trimethylation (H3K4me3) and gene transcription, which can regulate proliferation, apoptosis, and differentiation. However, little is known as to whether menin controls gene expression and cell proliferation and survival via regulating Polycomb group (PcG) protein complex/H3K27me3. Here we show that menin specifically represses transcription factor Paired box gene 2 (Pax2) through PcG-mediated H3K27me3 and Wilms tumor suppressor protein (WT1), a zinc finger domain-containing DNA-binding protein. Menin does not directly bind to the Pax2 locus, instead, it up-regulates WT1 expression. WT1 recruits PcG complex to the Pax2 promoter and represses expression of Pax2 through PcG-dependent H3K27me3. Moreover, WT1 also interacts with DNA methyltransferase 1 (DNMT1), and recruits DNMT1 to the Pax2 promoter, resulting in hypermethylation of CpG in the Pax2 promoter. Together, these studies have uncovered a novel epigenetic mechanism whereby menin regulates H3K27me3 and promoter DNA methylation via WT1 and suggest that WT1 protein plays an important, yet previously unappreciated role in regulating the function of the menin/PcG axis, H3K27 methylation, and DNA methylation, resulting in repression of gene transcription.

Resveratrol protects leukemic cells against cytotoxicity induced by proteasome inhibitors via induction of FOXO1 and p27Kip1

BackgroundIt was reported recently that resveratrol could sensitize a number of cancer cells to the antitumoral effects of some conventional chemotherapy drugs. The current study was designed to investigate whether resveratrol could sensitize leukemic cells to proteasome inhibitors.MethodsLeukemic cells were treated with MG132 alone or in combination with resveratrol. Cell viability was investigated using MTT assay, and induction of apoptosis and cell cycle distribution was measured using flow cytometry. Western blot and real-time RT-PCR were used to investigate the expression of FOXO1 and p27Kip1. CHIP was performed to investigate the binding of FOXO1 to the p27 Kip1 promoter.ResultsResveratrol strongly reduced cytotoxic activities of proteasome inhibitors against leukemic cells. MG132 in combination with resveratrol caused cell cycle blockade at G1/S transition via p27Kip1 accumulation. Knockdown of p27Kip1 using siRNA dramatically attenuated the protective effects of resveratrol on cytotoxic actions of proteasome inhibitors against leukemic cells. Resveratrol induced FOXO1 expression at the transcriptional level, while MG132 increased nuclear distribution of FOXO1. MG132 in combination with resveratrol caused synergistic induction of p27Kip1 through increased recruitment of FOXO1 on the p27Kip1 promoter.ConclusionsResveratrol may have the potential to negate the cytotoxic effects of proteasome inhibitors via regulation of FOXO1 transcriptional activity and accumulation of p27Kip1.

Tumor suppressor FOXO3 mediates signals from the EGF receptor to regulate proliferation of colonic cells

Epithelial proliferation, critical for homeostasis, healing, and colon cancer progression, is in part controlled by epidermal growth factor receptor (EGFR). Proliferation of colonic epithelia can be induced by Citrobacter rodentium infection, and we have demonstrated that activity of tumor suppressor FOXO3 was attenuated after this infection. Thus the aim of this study was to determine the contribution of FOXO3 in EGFR-dependent proliferation of intestinal epithelia and colon cancer cell lines. In this study we show that, during infection with C. rodentium, EGFR was significantly phosphorylated in colonic mucosa and Foxo3 deficiency in this model lead to an increased number of bromodeoxyuridine-positive cells. In vitro, in human colon cancer cells, increased expression and activation of EGFR was associated with proliferation that leads to FOXO3 phosphorylation (inactivation). Following EGFR activation, FOXO3 was phosphorylated (via phosphatidylinositol 3-kinase/Akt) and translocated to the cytosol where it was degraded. Moreover, inhibition of proliferation by overexpressing FOXO3 was not reversed by the EGFR signaling, implicating FOXO3 as one of the regulators downstream of EGFR. FOXO3 binding to the promoter of the cell cycle inhibitor p27kip1 was decreased by EGFR signaling, suggesting its role in EGFR-dependent proliferation. In conclusion, we show that proliferation in colonic epithelia and colon cancer cells, stimulated by EGFR, is mediated via loss of FOXO3 activity and speculate that FOXO3 may serve as a target in the development of new pharmacological treatments of proliferative diseases.