FOXM1 induces a global methylation signature that mimics the cancer epigenome in head and neck squamous cell carcinoma

The oncogene FOXM1 has been implicated in all major types of human cancer. We recently showed that aberrant FOXM1 expression causes stem cell compartment expansion resulting in the initiation of hyperplasia. We have previously shown that FOXM1 regulates HELLS, a SNF2/helicase involved in DNA methylation, implicating FOXM1 in epigenetic regulation. Here, we have demonstrated using primary normal human oral keratinocytes (NOK) that upregulation of FOXM1 suppressed the tumour suppressor gene p16^INK4A (CDKN2A) through promoter hypermethylation. Knockdown of HELLS using siRNA re-activated the mRNA expression of p16^INK4A and concomitant downregulation of two DNA methyltransferases DNMT1 and DNMT3B. The dose-dependent upregulation of endogenous FOXM1 (isoform B) expression during tumour progression across a panel of normal primary NOK strains (n = 8), dysplasias (n = 5) and head and neck squamous cell carcinoma (HNSCC) cell lines (n = 11) correlated positively with endogenous expressions of HELLS, BMI1, DNMT1 and DNMT3B and negatively with p16^INK4A and involucrin. Bisulfite modification and methylation-specific promoter analysis using absolute quantitative PCR (MS-qPCR) showed that upregulation of FOXM1 significantly induced p16^INK4A promoter hypermethylation (10-fold, P<0.05) in primary NOK cells. Using a non-bias genome-wide promoter methylation microarray profiling method, we revealed that aberrant FOXM1 expression in primary NOK induced a global hypomethylation pattern similar to that found in an HNSCC (SCC15) cell line. Following validation experiments using absolute qPCR, we have identified a set of differentially methylated genes, found to be inversely correlated with in vivo mRNA expression levels of clinical HNSCC tumour biopsy samples. This study provided the first evidence, using primary normal human cells and tumour tissues, that aberrant upregulation of FOXM1 orchestrated a DNA methylation signature that mimics the cancer methylome landscape, from which we have identified a unique FOXM1-induced epigenetic signature which may have clinical translational potentials as biomarkers for early cancer screening, diagnostic and/or therapeutic interventions.     

Epigallocatechin-3-gallate up-regulates tumor suppressor gene expression via a reactive oxygen species-dependent down-regulation of UHRF1

Ubiquitin-like containing PHD and Ring finger 1 (UHRF1) contributes to silencing of tumor suppressor genes by recruiting DNA methyltransferase 1 (DNMT1) to their hemi-methylated promoters. Conversely, demethylation of these promoters has been ascribed to the natural anti-cancer drug, epigallocatechin-3-gallate (EGCG). The aim of the present study was to investigate whether the UHRF1/DNMT1 pair is an important target of EGCG action. Here, we show that EGCG down-regulates UHRF1 and DNMT1 expression in Jurkat cells, with subsequent up-regulation of p73 and p16^INK4A genes. The down-regulation of UHRF1 is dependent upon the generation of reactive oxygen species by EGCG. Up-regulation of p16^INK4A is strongly correlated with decreased promoter binding by UHRF1. UHRF1 over-expression counteracted EGCG-induced G1-arrested cells, apoptosis, and up-regulation of p16^INK4A and p73. Mutants of the Set and Ring Associated (SRA) domain of UHRF1 were unable to down-regulate p16^INK4A and p73, either in the presence or absence of EGCG. Our results show that down-regulation of UHRF1 is upstream to many cellular events, including G1 cell arrest, up-regulation of tumor suppressor genes and apoptosis.     

p16INK4A Participates in a G1 Arrest Checkpoint in Response to DNA Damage

Members of the INK4 protein family specifically inhibit cyclin-dependent kinase 4 (cdk4) and cdk6-mediated phosphorylation of the retinoblastoma susceptibility gene product (Rb). p16^INK4A, a prototypic INK4 protein, has been identified as a tumor suppressor in many human cancers. Inactivation of p16^INK4A in tumors expressing wild-type Rb is thought to be required in order for many malignant cell types to enter S phase efficiently or to escape senescence. Here, we demonstrate another mechanism of tumor suppression by implicating p16^INK4A in a G₁ arrest checkpoint in response to DNA damage. Calu-1 non-small cell lung cancer cells, which retain Rb and lack p53, do not arrest in G₁ following DNA damage. However, engineered expression of p16^INK4A at levels compatible with cell proliferation restores a G₁ arrest checkpoint in response to treatment with γ-irradiation, topoisomerase I and II inhibitors, and cisplatin. A similar checkpoint can be demonstrated in p53^−/− fibroblasts that express p16^INK4A. DNA damage-induced G₁ arrest, which requires the expression of pocket proteins such as Rb, can be abrogated by overexpression of cdk4, kinase-inactive cdk4 variants capable of sequestering p16^INK4A, or a cdk4 variant incapable of binding p16^INK4A. After exposure to DNA-damaging agents, there was no change either in overall levels of p16^INK4A or in amounts of p16^INK4A found in complex with cdks 4 and 6. Nonetheless, p16^INK4A expression is required for the reduction in cdk4- and cdk6-mediated Rb kinase activity observed in response to DNA damage. During tumor progression, loss of p16^INK4A expression may be necessary for cells with wild-type Rb to bypass this G₁ arrest checkpoint and attain a fully transformed phenotype.     

Real-time in vivo imaging of p16Ink4a reveals cross talk with p53

Expression of the p16^Ink4a tumor suppressor gene, a sensor of oncogenic stress, is up-regulated by a variety of potentially oncogenic stimuli in cultured primary cells. However, because p16^Ink4a expression is also induced by tissue culture stress, physiological mechanisms regulating p16^Ink4a expression remain unclear. To eliminate any potential problems arising from tissue culture–imposed stress, we used bioluminescence imaging for noninvasive and real-time analysis of p16^Ink4a expression under various physiological conditions in living mice. In this study, we show that oncogenic insults such as ras activation provoke epigenetic derepression of p16^Ink4a expression through reduction of DNMT1 (DNA methyl transferase 1) levels as a DNA damage response in vivo. This pathway is accelerated in the absence of p53, indicating that p53 normally holds the p16^Ink4a response in check. These results unveil a backup tumor suppressor role for p16^Ink4a in the event of p53 inactivation, expanding our understanding of how p16^Ink4a expression is regulated in vivo.     

Association between P16INK4a Promoter Methylation and HNSCC: A Meta-Analysis of 21 Published Studies

Background

The p16^INK4a is an important tumor suppressor gene (TSG) and aberrant methylation of promoter is known to be a major inactivation mechanism of the tumor suppressor and tumor-related genes. Aberrant TSG methylation was considered an important epigenetic silencing mechanism in the progression of head and neck squamous cell carcinoma (HNSCC). However, some studies have reported differences in the methylation frequencies of P^16INK4a promoter between cancer and the corresponding control group. Therefore, we conducted a meta-analysis to better identify the association.

Methods

PubMed, Ovid, ISI Web of Science, and EMBASE were searched to identify eligible studies to evaluate the association of p16^INK4a promoter methylation and HNSCC. Odds ratio (ORs) and 95% confidence intervals (95%CI) were calculated to evaluate the strength of association between p16^INK4a promoter methylation and HNSCC.

Results

A total of twenty-one studies with 1155 cases and 1017 controls were included in the meta-analysis. The frequencies of p16^INK4a promoter methylation in the cancer group were significantly higher than those in the control group (cancer group: median: 46.67%, range = 7.84%-95.12%; control group: median: 18.37%, range = 0–83.33%; respectively). The pooled odds ratio was 3.37 (95%CI = 2.32–4.90) in the cancer group versus the corresponding control group under the random-effects model.

Conclusion

This meta-analysis of 21 published studies identified that aberrant methylation of p16^INK4a promoter was found to be significantly associated with HNSCC.     

Prognostic Significance of Overexpressed p16INK4a in Patients with Cervical Cancer: A Meta-Analysis

Background

p16^INK4a is a tumor suppressor protein which is induced in cells upon the interaction of high-risk HPV E7 with the retinoblastoma protein by a positive feedback loop, but cannot exert its suppressing effect. Previous reports suggested that p16^INK4a immunostaining allows precise identification of even small CIN or cervical cancer lesions in biopsies. The prognostic value of overexpressed p16^INK4a in cervical cancer has been evaluated for several years while the results remain controversial. We performed a systematic review and meta-analysis of studies assessing the clinical and prognostic significance of overexpression of p16^INK4a in cervical cancer.

Methods

Identification and review of publications assessing clinical or prognostic significance of p16^INK4a overexpression in cervical cancer until March 1, 2014. A meta-analysis was performed to clarify the association between p16^INK4a overexpression and clinical outcomes.

Results

A total of 15 publications met the criteria and comprised 1633 cases. Analysis of these data showed that p16^INK4a overexpression was not significantly associated with tumor TNM staging (I+II vs. III+IV) (OR = 0.75, 95% confidence interval [CI]: 0.35–1.63, P = 0.47), the tumor grade (G1+ G2 vs. G3) (OR = 0.78, 95% CI: 0.39–1.57, P = 0.49), the tumor size (<4 vs. ≥4 cm) (OR = 1.10, 95% CI: 0.45–2.69, P = 0.83), or vascular invasion (OR = 1.20, 95% CI: 0.69–2.08, P = 0.52). However, in the identified studies, overexpression of p16^INK4a was highly correlated with no lymph node metastasis (OR = 0.51, 95% CI: 0.28–0.95, P = 0.04), increased overall survival (relative risk [RR]: 0.42, 95% CI: 0.24–0.72, P = 0.002) and increased disease free survival (RR: 0.60, 95% CI: 0.44–0.82, P = 0.001).

Conclusions

This meta-analysis shows overexpression of p16^INK4a in cervical cancer is connected with increased overall and disease free survival and thus marks a better prognosis.     

Prognostic Value of Overexpressed p16INK4a in Vulvar Cancer: A Meta-Analysis

Objective

This study aimed to examine the prognostic value of overexpressed p16^INK4a in vulvar cancer. Although the tumor suppressor p16^INK4a has been shown to be of prognostic value in a wide variety of cancers and precancerous lesions, its role in the vulvar cancer is still unclear.

Methods

All publications in English language on the association between p16^INK4a and clinicopathological features of vulvar cancer were searched from Pubmed, Embase, and Web of Science, and those in Chinese language were identified manually and online from the China National Knowledge Infrastructure. Strict inclusion and exclusion criteria were followed. Odds ratios(ORs) or risk ratios(RRs) with 95% confidence intervals(CIs) were pooled to assess the strength of association. Publication bias was estimated using funnel plots and the Egger’s regression test.

Results

A total of 17 studies with 2309 patients were included. The p16^INK4a overexpression was found to correlate significantly with the lower International Federation of Gynecology and Obstetrics stage(I+II vs III+IV; OR = 0.60,95%CI:0.41–0.86,P = 0.006),negative lymph node metastasis(negative vs positive; OR = 0.61,95%CI:0.39–0.95,P = 0.029),patient’s age<55(OR = 0.54,95%CI:0.31–0.96,P = 0.034),human papillomavirus–positive status(OR = 0.01,95%CI:0.00–0.11,P<0.001),and higher overall survival(RR = 0.53,95%CI = 0.35–0.80,P = 0.003).

Conclusion

The p16^INK4a might be associated with a higher survival and indicates better prognosis of vulvar cancer.     

Targeting of 5-aza-2′-deoxycytidine residues by chromatin-associated DNMT1 induces proteasomal degradation of the free enzyme

5-Aza-2′-deoxycytidine (5-aza-dC) is a nucleoside analogue with cytotoxic and DNA demethylating effects. Here we show that 5-aza-dC induces the proteasomal degradation of free (non-chromatin bound) DNMT1 through a mechanism which is dependent on DNA synthesis and the targeting of incorporated 5-aza-dC residues by DNMT1 itself. Thus, 5-aza-dC induces Dnmt1 degradation in wild-type mouse ES cells, but not in Dnmt [3a^–/–, 3b^–/–] mouse ES cells which express Dnmt1 but lack DNA methylation (<0.7% of CpG methylated) and contain few hemi-methylated CpG sites, these being the preferred substrates for Dnmt1. We suggest that adducts formed between DNMT1 and 5-aza-dC molecules in DNA induce a ubiquitin-E3 ligase activity which preferentially targets free DNMT1 molecules for degradation by the proteasome. The proteasome inhibitor MG132 prevents DNMT1 degradation and reduces hypomethylation induced by 5-aza-dC.     

Loss of DNMT1o Disrupts Imprinted X Chromosome Inactivation and Accentuates Placental Defects in Females

The maintenance of key germline derived DNA methylation patterns during preimplantation development depends on stores of DNA cytosine methyltransferase-1o (DNMT1o) provided by the oocyte. Dnmt1o^mat−/− mouse embryos born to Dnmt1^Δ1o/Δ1o female mice lack DNMT1o protein and have disrupted genomic imprinting and associated phenotypic abnormalities. Here, we describe additional female-specific morphological abnormalities and DNA hypomethylation defects outside imprinted loci, restricted to extraembryonic tissue. Compared to male offspring, the placentae of female offspring of Dnmt1^Δ1o/Δ1o mothers displayed a higher incidence of genic and intergenic hypomethylation and more frequent and extreme placental dysmorphology. The majority of the affected loci were concentrated on the X chromosome and associated with aberrant biallelic expression, indicating that imprinted X-inactivation was perturbed. Hypomethylation of a key regulatory region of Xite within the X-inactivation center was present in female blastocysts shortly after the absence of methylation maintenance by DNMT1o at the 8-cell stage. The female preponderance of placental DNA hypomethylation associated with maternal DNMT1o deficiency provides evidence of additional roles beyond the maintenance of genomic imprints for DNA methylation events in the preimplantation embryo, including a role in imprinted X chromosome inactivation.     

A Novel Mutation in the Upstream Open Reading Frame of the CDKN1B Gene Causes a MEN4 Phenotype

The CDKN1B gene encodes the cyclin-dependent kinase inhibitor p27^KIP1, an atypical tumor suppressor playing a key role in cell cycle regulation, cell proliferation, and differentiation. Impaired p27^KIP1 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 p27^KIP1 expression. Our findings demonstrate that, in addition to the previously described mechanisms leading to reduced p27^KIP1 activity, such as degradation via the ubiquitin/proteasome pathway or non-covalent sequestration, p27^KIP1 activity can also be modulated by an uORF and mutations affecting uORF could change p27^KIP1 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.     

The 5′-flanking region of the E1 α form of the murine p16 (MTS1) gene

We have PCR-amplified and sequenced the immediate (841 bp) 5′-flanking region of murine p16^INK4a (MTS1, CDKN2) tumor suppressor gene. Comparing to recently published 5'-flanking region of the human α form of p16^INK4a, homologies were found in several regions of murine p16^INK4a-α putative promoter sequence.     

Senescence delay and repression of p16INK4a by Lsh via recruitment of histone deacetylases in human diploid fibroblasts

Lymphoid specific helicase (Lsh) belongs to the family of SNF2/helicases. Disruption of Lsh leads to developmental growth retardation and premature aging in mice. However, the specific effect of Lsh on human cellular senescence remains unknown. Herein, we report that Lsh overexpression delays cell senescence by silencing p16^INK4a in human fibroblasts. The patterns of p16^INK4a and Lsh expression during cell senescence present the inverse correlation. We also find that Lsh requires histone deacetylase (HDAC) activity to repress p16^INK4a and treatment with trichostatin A (TSA) is sufficient to block the repressor effect of Lsh. Moreover, overexpression of Lsh is correlated with deacetylation of histone H3 at the p16 promoter, and TSA treatment in Lsh-expressing cells reverses the acetylation status of histones. Additionally, we demonstrate an interaction between Lsh, histone deacetylase 1 (HDAC1) and HDAC2 in vivo. Furthermore, we demonstrate that Lsh interacts in vivo with the p16 promoter and recruits HDAC1. Our data suggest that Lsh represses endogenous p16^INK4a expression by recruiting HDAC to establish a repressive chromatin structure at the p16^INK4a promoter, which in turn delays cell senescence.     

The OGF-OGFr axis utilizes the p16INK4a and p21WAF1/CIP1 pathways to restrict normal cell proliferation

Opioid growth factor (OGF) is an endogenous opioid peptide ([Met⁵]enkephalin) that interacts with the OGF receptor (OGFr) and serves as a tonically active negative growth factor in cell proliferation of normal cells. To clarify the mechanism by which OGF inhibits cell replication in normal cells, we investigated the effect of the OGF–OGFr axis on cell cycle activity in human umbilical vein endothelial cells (HUVECs) and human epidermal keratinocytes (NHEKs). OGF markedly depressed cell proliferation of both cell lines by up to 40% of sterile water controls. Peptide treatment induced cyclin-dependent kinase inhibitor (CKI) p16^INK4a protein expression and p21^WAF1/CIP1 protein expression in HUVECs and NHEKs, but had no effect on p15, p18, p19, or p27 protein expression in either cell type. Inhibition of either p16^INK4a or p21^WAF1/CIP1 activation by specific siRNAs blocked OGF inhibitory action. Human dermal fibroblasts and mesenchymal stem cells also showed a similar dependence of OGF action on p16^INK4a and p21^WAF1/CIP1. Collectively, these results indicate that both p16^INK4a and p21^WAF1/CIP1 are required for the OGF–OGFr axis to inhibit cell proliferation in normal cells.