p16INK4a and p14ARF methylation as a potential biomarker for human bladder cancer

Promoter hypermethylation is one of the putative mechanisms underlying the inactivation of negative cell-cycle regulators. We examined whether the methylation status of p16(INK4a) and p14(ARF), genes located upstream of the RB and p53 pathway, is a useful biomarker for the staging, clinical outcome, and prognosis of human bladder cancer. Using methylation-specific PCR (MSP), we examined the methylation status of p16(INK4a) and p14(ARF) in 64 samples from 45 bladder cancer patients (34 males, 11 females). In 19 patients with recurrent bladder cancer, we examined paired tissue samples from their primary and recurrent tumors. The methylation status of representative samples was confirmed by bisulfite DNA sequencing analysis. The median follow-up duration was 34.3 months (range 27.0-100.1 months). The methylation rate for p16(INK4a) and p14(ARF) was 17.8% and 31.1%, respectively, in the 45 patients. The incidence of p16(INKa) and p14(ARF) methylation was significantly higher in patients with invasive (>or=pT2) than superficial bladder cancer (pT1) (p=0.006 and p=0.001, respectively). No MSP bands for p16(INK4a) and p14(ARF) were detected in the 8 patients with superficial, non-recurrent tumors. In 19 patients with tumor recurrence, the p16(INK4a) and p14(ARF) methylation status of the primary and recurrent tumors was similar. Of the 22 patients who had undergone cystectomy, 8 (36.4%) manifested p16(INKa) methylation; p16(INK4a) was not methylated in 23 patients without cystectomy (p=0.002). Kaplan-Meier analysis revealed that patients with p14(ARF) methylation had a significantly poorer prognosis than those without (p=0.029). This is the first study indicating that MSP analysis of p16(INK4a) and p14(ARF) genes is a useful biomarker for the pathological stage, clinical outcome, and prognosis of patients with bladder cancer.     

Identification and sequencing of the Syrian Golden hamster (Mesocricetus auratus) p16(INK4a) and p15(INK4b) cDNAs and their homozygous gene deletion in cheek pouch and pancreatic tumor cells.

Previous studies have shown that the p16(INK4a) tumor suppressor gene is inactivated in up to 98% of human pancreatic cancer specimens and 83% of oral squamous cell carcinomas. Inactivation of the related p15(INK4b) gene has also been identified in a number of tumors and cell lines, however, its role as an independent tumor suppressor remains to be elucidated. Chemically-induced tumors in the Syrian Golden hamster (Mesocricetus auratus) have been shown to be excellent representative models for the comparative development and progression of a number of human malignancies. The purpose of this study was to determine the importance of the p16(INK4a) and p15(INK4b) genes in two experimental hamster models for human pancreatic and oral carcinogenesis. First, hamster p16(INK4a) and p15(INK4b) cDNAs were cloned and sequenced. The hamster p16(INK4a) cDNA open reading frame (ORF) shares 78%, 80%, and 81% identity with the human, mouse, and rat p16(INK4a) sequences, respectively. Similarly, the hamster p15(INK4b) cDNA ORF shares 82% and 89% sequence identity with human and mouse p15(INK4b), respectively. Second, a deletion analysis of hamster p16(INK4a) and p15(INK4b) genes was performed for several tumorigenic and non-tumorigenic hamster cell lines and revealed that both p16(INK4a) and p15(INK4b) were homozygously deleted in a cheek pouch carcinoma cell line (HCPC) and two pancreatic adenocarcinoma cell lines (KL5B, H2T), but not in tissue matched, non-tumorigenic cheek pouch (POT2) or pancreatic (KL5N) cell lines. These data strongly suggest that homozygous deletion of the p16(INK4a) and p15(INK4b) genes plays a prominent role in hamster pancreatic and oral tumorigenesis, as has been well established in correlative studies in comparable human tumors. Furthermore, this study supports the comparative importance of the hamster pancreatic and cheek pouch models of carcinogenesis in subsequent mechanistic-, therapeutic-, and preventive-based studies aimed at providing important translational data applicable to pancreatic adenocarcinoma and oral squamous cell carcinoma in humans.     

Design and characterization of a hyperstable p16INK4a that restores Cdk4 binding activity when combined with oncogenic mutations

Cyclin-dependent kinase inhibitor p16(INK4a) is the founding member of the INK4 family of tumor suppressors capable of arresting mammalian cell division. Missense mutations in the p16(INK4a) gene (INK4a/CDKN2A/MTS1) are strongly linked to several types of human cancer. These mutations are evenly distributed throughout this small, ankyrin repeat protein and the majority of them disrupt the native secondary and/or tertiary structure, leading to protein unfolding, aggregation and loss of function. We report here the use of multiple stabilizing substitutions to increase the stability of p16(INK4a) and furthermore, to restore Cdk4 binding activity of several defective, cancer-related mutant proteins. Stabilizing substitutions were predicted using four different techniques. The three most effective substitutions were combined to create a hyperstable p16(INK4a) variant that is 1.4 kcal/mol more stable than wild-type. This engineered construct is monomeric in solution with wild-type-like secondary and tertiary structure and cyclin-dependent kinase 4 binding activity. Interestingly, these hyperstable substitutions, when combined with oncogenic mutations R24P, P81L or V126D, can significantly restore Cdk4 binding activity, despite the divergent features of each destabilizing mutation. Extensive biophysical studies indicate that the hyperstable substitutions enhance the binding activity of mutant p16 through several different mechanisms, including an increased amount of secondary structure and thermostability, reduction in exposed hydrophobic surface(s) and/or a reduced tendency to aggregate. This apparent global suppressor effect suggests that increasing the thermodynamic stability of p16 can be used as a general strategy to restore the biological activity to defective mutants of this important tumor suppressor protein.     

In silico analysis of structural and functional consequences in p16INK4A by deleterious nsSNPs associated CDKN2A gene in malignant melanoma

In this study, we identified the most deleterious nsSNP in CDKN2A gene through structural and functional properties of its protein (p16INK4A) and investigated its binding affinity with cdk6. Out of 118 SNPs, 14 are nsSNPs in the coding region and 17 SNPs were found in the untranslated region (UTR). FastSNP suggested that 7 SNPs in the 5' UTR might change the protein expression levels. Sixty-four percent of nsSNPs are found to be damaged in PolyPhen server among the 14 nsSNPs investigated. With this effort, we modeled the mutant p16INK4A proteins based on these deleterious nsSNPs, out of which three nsSNPs associated p16INK4A had RMSD values of greater than 3.00 A with native protein. From a comparison of total energy of these three mutant proteins, we identified that the major mutation is from Aspartic acid to Tyrosine at the residue position of 84 of p16INK4A. Further, we compared the binding efficiency of both native and mutant p16INK4A with cdk6. We found that mutant p16INK4A has less binding affinity with cdk6 compared to native type. This is due to ten hydrogen bonds and eight salt bridges which exist between the native type and cdk6, whereas the mutant type makes only nine hydrogen bonds and five salt bridges with cdk6. Based on our investigation, we propose that the SNP with the ID rs11552822 could be the most deleterious nsSNP in CDKN2A gene, causing malignant melanoma, as it was well correlated with experimental studies carried out elsewhere.     

Promoter hypermethylation of p16INK4A, p14ARF, CyclinD2 and Slit2 in serum and tumor DNA from breast cancer patients

Epigenetic mechanisms such as DNA methylation play important role in cancer. Epigenetic alterations involved in the onset and progression of breast cancer may serve as biomarkers for early detection and prediction of disease prognosis. Furthermore, using body fluids such as serum offers a non-invasive method to procure multiple samples for biomarker analyses. The aim of this study is to determine the correlation between methylation status of multiple cancer genes, p16(INK4A), p14(ARF), Cyclin D2 and Slit2 in invasive ductal carcinoma of the breast and paired serum DNA and clinicopathological parameters. Of the 36 breast cancer patients investigated, 31 (86%) tumors and 30 (83%) paired sera showed methylation of at least one of these 4 genes. Methylation frequencies varied from 27% for CyclinD2, 44% for p16(INK4A), 47% for p14(ARF) to 58% for Slit2. There was concordance between DNA methylation in tumor and paired serum DNA of each gene. This study underscores the potential utility of DNA methylation based screening of serum as a surrogate marker for tumor DNA methylation status of these genes in breast cancer. Further, expression profile of p16(INK4A) could be linked to epigenetic events, thus suggesting this pathway as a potential target for therapeutic strategies based on reversal of epigenetic silencing.     

p16INK4A Positively Regulates p21WAF1 Expression by suppressing AUF1-dependent mRNA decay

Background

p16^INK4a and p21^WAF1 are two independent cyclin-dependent kinase inhibitors encoded by the CDKN2A and CDKN1A genes, respectively. p16^INK4a and p21^WAF1 are similarly involved in various anti-cancer processes, including the regulation of the critical G1 to S phase transition of the cell cycle, senescence and apoptosis. Therefore, we sought to elucidate the molecular mechanisms underlying the link between these two important tumor suppressor proteins.

Methodology/Principal Findings

We have shown here that the p16^INK4a protein positively controls the expression of p21^WAF1 in both human and mouse cells. p16^INK4a stabilizes the CDKN1A mRNA through negative regulation of the mRNA decay-promoting AUF1 protein. Immunoprecipitation of AUF1-associated RNAs followed by quantitative RT-PCR indicated that endogenous AUF1 binds to the CDKN1A mRNA in a p16^INK4A-dependent manner. Furthermore, while AUF1 down-regulation increased the expression level of the CDKN1A mRNA, the concurrent knockdown of AUF1 and CDKN2A, using specific silencing RNAs, restored the normal expression of the gene. Moreover, we used EGFP reporter fused to the CDKN2A AU-rich element (ARE) to demonstrate that p16^INK4A regulation of the CDKN1A mRNA is AUF1- and ARE-dependent. Furthermore, ectopic expression of p16^INK4A in p16^INK4A-deficient breast epithelial MCF-10A cells significantly increased the level of p21^WAF1, with no effect on cell proliferation. In addition, we have shown direct correlation between p16^INK4a and p21^WAF1 levels in various cancer cell lines.

Conclusion/Significance

These findings show that p16^INK4a stabilizes the CDKN1A mRNA in an AUF1-dependent manner, and further confirm the presence of a direct link between the 2 important cancer-related pathways, pRB/p16^INK4A and p14^ARF/p53/p21^WAF1.     

Identification and characterization of a novel protein ISOC2 that interacts with p16INK4a

p16(INK4a) is a multiple tumor suppressor, playing an important role in proliferation and tumorigenesis. To screen the p16(INK4a)-associated proteins, we performed a yeast two-hybrid assay and identified a novel protein isochorismatase domain containing 2 (ISOC2). ISOC2 conserves in different species, and encodes 205 and 210 amino acids in human and mouse, respectively. The expression of ISOC2 in mouse is universal but predominantly in uterus, stomach, and urinary tract system. Interaction between ISOC2 and p16(INK4a) was verified using in vitro pull-down assays and in vivo co-immunoprecipitation. Confocal microscopy studies using green and cyan fluorescent fusion proteins determined that ISOC2 co-localizes with p16(INK4a). Over-expressed ISOC2 is able to inhibit p16(INK4a) in dose-dependent manner. Our data indicated that ISOC2 is a novel functional protein, which is able to bind and co-localize with a tumor suppressor gene p16(INK4a). Over-expressed ISOC2 inhibits the expression of p16(INK4a), suggesting that this novel gene may play a role during the tumor development by interacting with p16(INK4a).     

Analysis of p16 expression and allelic imbalance / loss of heterozygosity of 9p21 in cutaneous squamous cell carcinomas

Deletions of the short arm of chromosome 9 have been reported in different types of malignancies. This chromosomal region contains a number of known tumour suppressor genes, including the p16INK4A (CDKN2A), p15INK4B and MTAP tumour suppressor genes located at 9p21. In this study twenty-two paraffin embedded invasive cutaneous SCC were examined for allelic imbalance/ loss of heterozygosity (AI/LOH) of the 9p region (in particular 9p21), and for p16 protein expression. DNA was isolated from microdissected sections of normal and tumour cells and analysed for AI/LOH by using six fluorescently labelled microsatellite markers that map to the 9p region. P16 protein expression was examined by immunohistochemistry. At each of the six microsatellite markers the majority of SCC analysed showed AI/LOH. Overall both AI/LOH within the CDKN2A locus and absence of p16 protein expression were frequent among the cutaneous SCC analysed, suggesting that p16 inactivation may play a role in cutaneous SCC development. The majority of the SCC analysed also had AI/LOH of the marker within the MTAP gene, and at markers flanking the CDKN2A gene; thus further investigation as to a possible role for these genes in the development of cutaneous SCC is warranted.     

Inactivation of p16INK4a in Primary Tumors and Cell Lines of Head and Neck Squamous Cell Carcinoma

Inactivation of the p16^INK4a gene by mutation and deletion is common in head and neck squamous cell carcinoma (HNSCC). The present study demonstrates that hypermethylation of the 5 CpG islands can serve as an alternative mechanism for the inactivation of the p16^INK4a gene in this tumor. We studied 11 HNSCC cell lines and 17 oral squamous cell carcinoma (OSCC) primary tumors for p16^INK4a gene status by protein/mRNA and DNA genetic/epigenetic analyses to determine the incidence of its inactivation. Our study indicates that: (1) inactivation of p16 protein is frequent in HNSCC cell lines (6/11, 54.5%) and OSCC primary tumors (15/17, 88.2%), (2) inactivation of p16^INK4a protein is commonly associated with the presence of gene alteration such as mutation, homozygous deletion and especially aberrant methylation, and (3) genomic sequencing of bisulfite-modified DNA shows that the carcinoma develops a heterogeneous pattern of hypermethylation.     

Adenovirus-mediated overexpression of p15INK4B inhibits human glioma cell growth, induces replicative senescence, and inhibits telomerase activity similarly to p16INK4A.

The genes encoding the cyclin-dependent kinase inhibitors p16INK4A (CDKN2A) and p15INK4B (CDKN2B) are frequently homozygously deleted in a variety of tumor cell lines and primary tumors, including glioblastomas in which 40-50% of primary tumors display homozygous deletions of these two loci. Although the role of p16 as a tumor suppressor has been well documented, it has remained less well studied whether p15 plays a similar growth-suppressing role. Here, we have used replication-defective recombinant adenoviruses to compare the effects of expressing wild-type p16 and p15 in glioma cell lines. After infection, high levels of p16 and p15 were observed in two human glioma cell lines (U251 MG and U373 MG). Both inhibitors were found in complex with CDK4 and CDK6. Expression of p16 and p15 had indistinguishable effects on U251 MG, which has homozygous deletion of CDKN2A and CDKN2B, but a wild-type retinoblastoma (RB) gene. Cells were growth-arrested, showed no increased apoptosis, and displayed a markedly altered cellular morphology and repression of telomerase activity. Transduced cells became enlarged and flattened and expressed senescence-associated beta-galactosidase, thus fulfilling criteria for replicative senescence. In contrast, the growth and morphology of U373 MG, which expresses p16 and p15 endogenously, but undetectable levels of RB protein, were not affected by exogenous overexpression of either inhibitor. Thus, we conclude that overexpression of p15 has a similar ability to inhibit cell proliferation, to cause replicative senescence, and to inhibit telomerase activity as p16 in glioma cells with an intact RB protein pathway.     

p16INK4a基因的功能及其调控

p16^INK4a蛋白能抑制CDK4和CDK6的活性,使pRb处于非磷酸化或低磷酸化状态而能与转录因子E2Fs结合,从而抑制DNA 的合成,阻止细胞由G1期进入S期.p16^INK4a的表达受Ets1和Ets2的正调控,受Bmi-1的负调控.p16^INK4a基因缺失、突变、甲基化、RNA剪接加工错误可导致细胞周期失控和癌变.应用p16^INK4a对某些肿瘤进行基因治疗的研究正在进行中.     

Induction of apoptosis in p16INK4A mutant cell lines by adenovirus-mediated overexpression of p16INK4A protein

The tumor suppressor gene p16INK4A is a cyclin-dependent kinase inhibitor (CDKI) and an important cell cycle regulator. We have previously constructed a recombinant adenovirus which expresses p16 (Adp16) and shown that infection in a variety of human tumor cell lines with this recombinant virus results in high levels of p16INK4A protein expression resulting in cell cycle arrest and loss of cyclin-cdk activity. Furthermore, adenoviral-mediated overexpression of wild-type p16INK4A is more toxic in cancer cells which express mutant forms of p16INK4A compared to cancer cell lines containing endogenous wild-type p16. TUNEL assay and DAPI staining following infection of MDA-MB 231 breast cancer cells with Adp16 indicate that p16INK4A-mediated cytotoxicity was associated with apoptosis. This is supported by studies demonstrating a decrease in cpp32 and cyclinB1 protein levels and induction of poly (ADP-ribose) polymerase (PARP) cleavage following infection of MDA-MB-231 cells with Adp16. These results suggest that gene therapy using Adp16 may be a promising treatment option for human cancers containing alterations in p16 expression.     

p16INK4a-induced senescence is disabled by melanoma-associated mutations

The p16(INK4a)-Rb tumour suppressor pathway is required for the initiation and maintenance of cellular senescence, a state of permanent growth arrest that acts as a natural barrier against cancer progression. Senescence can be overcome if the pathway is not fully engaged, and this may occur when p16(INK4a) is inactivated. p16(INK4a) is frequently altered in human cancer and germline mutations affecting p16(INK4a) have been linked to melanoma susceptibility. To characterize the functions of melanoma-associated p16(INK4a) mutations, in terms of promoting proliferative arrest and initiating senescence, we utilized an inducible expression system in a melanoma cell model. We show that wild-type p16(INK4a) promotes rapid cell cycle arrest that leads to a senescence programme characterized by the appearance of chromatin foci, activation of acidic beta-galactosidase activity, p53 independence and Rb dependence. Accumulation of wild-type p16(INK4a) also promoted cell enlargement and extensive vacuolization independent of Rb status. In contrast, the highly penetrant p16(INK4a) variants, R24P and A36P failed to arrest cell proliferation and did not initiate senescence. We also show that overexpression of CDK4, or its homologue CDK6, but not the downstream kinase, CDK2, inhibited the ability of wild-type p16(INK4a) to promote cell cycle arrest and senescence. Our data provide the first evidence that p16(INK4a) can initiate a CDK4/6-dependent autonomous senescence programme that is disabled by inherited melanoma-associated mutations.     

Direct evidence from siRNA-directed "knock down" that p16(INK4a) is required for human fibroblast senescence and for limiting ras-induced epithelial cell proliferation

The selective pressure for disruption of the cyclin-dependent kinase inhibitor p16(INK4a) in human cancer has been postulated to reflect its role in mediating growth arrest, both in response to telomere erosion (replicative senescence) and to oncogene-induced and other "stress" signals. Given the known species-specific differences in regulation of senescence, we have tested this hypothesis in human, as opposed to rodent, cells by designing a small interfering RNA (siRNA) to knock down p16(INK4a) expression. Transfection of this siRNA into late-passage normal human diploid fibroblasts allowed at least temporary escape from entry into replicative senescence. Furthermore, in our in vitro model of early-stage, RAS-induced thyroid tumorigenesis, sequential transfections with this siRNA allowed outgrowth of small clusters of proliferating epithelial cells, consistent with escape from the spontaneous "senescence", which normally curtails their proliferative response to mutant RAS. These data provide the first direct evidence that p16(INK4a) is necessary for the initiation of both telomere-dependent and telomere-independent senescence in human cells.     

H3K27 Trimethylation Is an Early Epigenetic Event of p16INK4a Silencing for Regaining Tumorigenesis in Fusion Reprogrammed Hepatoma Cells

Stable epigenetic silencing of p16^INK4a is a common event in hepatocellular carcinoma (HCC) cells, which is associated with abnormal cell proliferation and liberation from cell cycle arrest. Understanding the early epigenetic events in silencing p16^INK4a expression may illuminate a prognostic strategy to block HCC development. Toward this end, we created a reprogram cell model by the fusion mouse HCC cells with mouse embryonic stem cells, in which the ES-Hepa hybrids forfeited HCC cell characteristics along with reactivation of the silenced p16^INK4a. HCC characteristics, in terms of gene expression pattern and tumorigenic potential, was restored upon induced differentiation of these reprogrammed ES-Hepa hybrids. The histone methylation pattern relative to p16^INK4a silencing during differentiation of the ES-Hepa hybrids was analyzed. H3K27 trimethylation at the p16^INK4a promoter region, occurring in the early onset of p16^INK4a silencing, was followed by H3K9 dimethylation at later stages. During the induced differentiation of the ES-Hepa hybrids, H3K4 di- and trimethylations were maintained at high levels during the silencing of p16^INK4a, strongly suggesting that H3K4 methylation events did not cause the silencing of p16^INK4a. Our results suggested that the enrichment of H3K27 trimethylation, independent of H3K9 dimethylation, trimethylation, and DNA methylation, was an early event in the silencing of p16^INK4a during the tumor development. This unique chromatin pattern may be a heritable marker of epigenetic regulation for p16^INK4a silencing during the developmental process of hepatocellular carcinogenesis.     

An evaluation of p16INK4a expression in cervical intraepithelial neoplasia specimens, including women with HIV-1

Although several studies have evaluated the role of p16INK4a as a diagnostic marker of cervical intraepithelial neoplasia (CIN) and its association with disease progression, studies regarding the role of p16INK4a in human immunodeficiency virus (HIV)-infected patients remain scarce. The present study was designed to determine the potential utility of p16INK4a as a diagnostic marker for CIN and invasive cervical cancer in HIV-positive and negative cervical specimens. An immunohistochemical analysis of p16INK4a was performed in 326 cervical tissue microarray specimens. Performance indicators were calculated and compared using receiving operating characteristics curve (ROC)/area under the curve. In HIV-1-negative women, the percentage of cells that was positive for p16INK4a expression was significantly correlated with the severity of CIN (p < 0.0001). A ROC curve with a cut-off value of 55.28% resulted in a sensitivity of 89%, a specificity of 81%, a positive predictive value of 91% and a negative predictive value of 78%. HIV-seropositive women exhibited decreased expression of p16INK4a in CIN2-3 specimens compared with HIV-negative specimens (p = 0.031). The ROC data underscore the potential utility of p16INK4a under defined conditions as a diagnostic marker for CIN 2-3 staging and invasive cervical cancer. HIV-1 infection, however, is associated with relatively reduced p16INK4a expression in CIN 2-3.