ZNF714 Supports Pro-Oncogenic Features in Lung Cancer Cells.

Despite the ongoing progress in diagnosis and treatments, cancer remains a threat to more than one-third of the human population. The emerging data indicate that many Krüppel-associated box zinc finger proteins (KRAB-ZNF) belonging to a large gene family may be involved in carcinogenesis. Our previous study identified Zinc Finger Protein 714 (ZNF714), a KRAB-ZNF gene of unknown function, as being commonly overexpressed in many tumors, pointing to its hypothetical oncogenic role. Here, we harnessed The Cancer Genome Atlas (TCGA)-centered databases and performed functional studies with transcriptomic and methylomic profiling to explore ZNF714 function in cancer. Our pan-cancer analyses confirmed frequent ZNF714 overexpression in multiple tumors, possibly due to regional amplification, promoter hypomethylation, and Nuclear Transcription Factor Y Subunit Beta (NFYB) signaling. We also showed that ZNF714 expression correlates with tumor immunosuppressive features. The in vitro studies indicated that ZNF714 expression positively associates with proliferation, migration, and invasion. The transcriptomic analysis of ZNF714 knocked-down cells demonstrated deregulation of cell adhesion, migration, proliferation, apoptosis, and differentiation. Importantly, we provided evidence that ZNF714 negatively regulates the expression of several known TSGs indirectly via promoter methylation. However, as ZNF714 did not show nuclear localization in our research model, the regulatory mechanisms exerted by ZNF714 require further investigation. In conclusion, our results reveal, for the first time, that ZNF714 may support pro-oncogenic features in lung cancer cells.

ZNF643/ZFP69B Exerts Oncogenic Properties and Associates with Cell Adhesion and Immune Processes.

The global cancer burden remains high; thus, a better understanding of the molecular mechanisms driving carcinogenesis is needed to improve current prevention and treatment options. We previously detected the ZNF643/ZFP69B gene upregulated in multiple tumors, and we speculated it may play a role in tumor biology. To test this hypothesis, we employed TCGA-centered databases to correlate ZNF643 status with various clinicopathological parameters. We also performed RNA-seq analysis and in vitro studies assessing cancer cell phenotypes, and we searched for ZNF643-bound genomic loci. Our data indicated higher levels of ZNF643 in most analyzed tumors compared to normal samples, possibly due to copy number variations. ZNF643 mRNA correlated with diverse molecular and immune subtypes and clinicopathological features (tumor stage, grade, patient survival). RNA-seq analysis revealed that ZNF643 silencing triggers the deregulation of the genes implicated in various cancer-related processes, such as growth, adhesion, and immune system. Moreover, we observed that ZNF643 positively influences cell cycle, migration, and invasion. Finally, our ChIP-seq analysis indicated that the genes associated with ZNF643 binding are linked to adhesion and immune signaling. In conclusion, our data confirm the oncogenic properties of ZNF643 and pinpoint its impact on cell adhesion and immune processes.

KRAB-ZFPs and cancer stem cells identity.

Studies on carcinogenesis continue to provide new information about different disease-related processes. Among others, much research has focused on the involvement of cancer stem cells (CSCs) in tumor initiation and progression. Studying the similarities and differences between CSCs and physiological stem cells (SCs) allows for a better understanding of cancer biology. Recently, it was shown that stem cell identity is partially governed by the Krϋppel-associated box domain zinc finger proteins (KRAB-ZFPs), the biggest family of transcription regulators. Several KRAB-ZFP factors exert a known effect in tumor cells, acting as tumor suppressor genes (TSGs) or oncogenes, yet their role in CSCs is still poorly characterized. Here, we review recent studies regarding the influence of KRAB-ZFPs and their cofactor protein TRIM28 on CSCs phenotype, stemness features, migration and invasion potential, metastasis, and expression of parental markers.

Zinc Finger Proteins in Head and Neck Squamous Cell Carcinomas: ZNF540 May Serve as a Biomarker.

Head and neck squamous cell carcinoma (HNSCC) is one of the ten most common cancers. Most cancer cases originate from alcohol and tobacco consumption. However, studies have demonstrated that human papillomavirus (HPV) infection, particularly HPV-16, may also significantly influence disease progression. The KRAB-ZNF family of genes is involved in epigenetic suppression, and its involvement in carcinogenesis is the subject of extensive studies. The available literature data demonstrate that they may play different roles, both as tumor suppressors and oncogenes. In this study, six ZNF genes, ZFP28, ZNF132, ZNF418, ZNF426, ZNF540, and ZNF880, were tested using several in silico approaches based on the TCGA and GEO datasets. Our analyses indicate that the expression of the analyzed ZNFs was significantly downregulated in tumor tissues and depended on tumor localization. The expression levels of ZNFs differed between HPV-positive vs. HPV-negative patients depending on the clinical-pathological parameters. More specifically, the patients with higher levels of ZNF418 and ZNF540 showed better survival rates than those with a lower expression. In addition, the level of ZNF540 expression in HPV-positive (HPV(+)) patients was higher than in HPV-negative (HPV(-)) patients (p < 0.0001) and was associated with better overall survival (OS). In conclusion, we demonstrate that ZNF540 expression highly correlates with HPV infection, which renders ZNF540 a potential biomarker for HNSCC prognosis and treatment.

Causes, effects, and clinical implications of perturbed patterns within the cancer epigenome.

Somatic mutations accumulating over a patient's lifetime are well-defined causative factors that fuel carcinogenesis. It is now clear, however, that epigenomic signature is also largely perturbed in many malignancies. These alterations support the transcriptional program crucial for the acquisition and maintenance of cancer hallmarks. Epigenetic instability may arise due to the genetic mutations or transcriptional deregulation of the proteins implicated in epigenetic signaling. Moreover, external stimulation and physiological aging may also participate in this phenomenon. The epigenomic signature is frequently associated with a cell of origin, as well as with tumor stage and differentiation, which all reflect its high heterogeneity across and within various tumors. Here, we will overview the current understanding of the causes and effects of the altered and heterogeneous epigenomic landscape in cancer. We will focus mainly on DNA methylation and post-translational histone modifications as the key regulatory epigenetic signaling marks. In addition, we will describe how this knowledge is translated into the clinic. We will particularly concentrate on the applicability of epigenetic alterations as biomarkers for improved diagnosis, prognosis, and prediction. Finally, we will also review current developments regarding epi-drug usage in clinical and experimental settings.

Disruption of RING and PHD Domains of TRIM28 Evokes Differentiation in Human iPSCs.

TRIM28, a multi-domain protein, is crucial in the development of mouse embryos and the maintenance of embryonic stem cells' (ESC) self-renewal potential. As the epigenetic factor modulating chromatin structure, TRIM28 regulates the expression of numerous genes and is associated with progression and poor prognosis in many types of cancer. Because of many similarities between highly dedifferentiated cancer cells and normal pluripotent stem cells, we applied human induced pluripotent stem cells (hiPSC) as a model for stemness studies. For the first time in hiPSC, we analyzed the function of individual TRIM28 domains. Here we demonstrate the essential role of a really interesting new gene (RING) domain and plant homeodomain (PHD) in regulating pluripotency maintenance and self-renewal capacity of hiPSC. Our data indicate that mutation within the RING or PHD domain leads to the loss of stem cell phenotypes and downregulation of the FGF signaling. Moreover, impairment of RING or PHD domain results in decreased proliferation and impedes embryoid body formation. In opposition to previous data indicating the impact of phosphorylation on TRIM28 function, our data suggest that TRIM28 phosphorylation does not significantly affect the pluripotency and self-renewal maintenance of hiPSC. Of note, iPSC with disrupted RING and PHD functions display downregulation of genes associated with tumor metastasis, which are considered important targets in cancer treatment. Our data suggest the potential use of RING and PHD domains of TRIM28 as targets in cancer therapy.

KRAB-ZFP Transcriptional Regulators Acting as Oncogenes and Tumor Suppressors: An Overview.

Krüppel-associated box zinc finger proteins (KRAB-ZFPs) constitute the largest family of transcriptional factors exerting co-repressor functions in mammalian cells. In general, KRAB-ZFPs have a dual structure. They may bind to specific DNA sequences via zinc finger motifs and recruit a repressive complex through the KRAB domain. Such a complex mediates histone deacetylation, trimethylation of histone 3 at lysine 9 (H3K9me3), and subsequent heterochromatization. Nevertheless, apart from their repressive role, KRAB-ZFPs may also co-activate gene transcription, likely through interaction with other factors implicated in transcriptional control. KRAB-ZFPs play essential roles in various biological processes, including development, imprinting, retroelement silencing, and carcinogenesis. Cancer cells possess multiple genomic, epigenomic, and transcriptomic aberrations. A growing number of data indicates that the expression of many KRAB-ZFPs is altered in several tumor types, in which they may act as oncogenes or tumor suppressors. Hereby, we review the available literature describing the oncogenic and suppressive roles of various KRAB-ZFPs in cancer. We focused on their association with the clinicopathological features and treatment response, as well as their influence on the cancer cell phenotype. Moreover, we summarized the identified upstream and downstream molecular mechanisms that may govern the functioning of KRAB-ZFPs in a cancer setting.

CRISPR/Cas9 in Cancer Immunotherapy: Animal Models and Human Clinical Trials.

Even though chemotherapy and immunotherapy emerged to limit continual and unregulated proliferation of cancer cells, currently available therapeutic agents are associated with high toxicity levels and low success rates. Additionally, ongoing multi-targeted therapies are limited only for few carcinogenesis pathways, due to continually emerging and evolving mutations of proto-oncogenes and tumor-suppressive genes. CRISPR/Cas9, as a specific gene-editing tool, is used to correct causative mutations with minimal toxicity, but is also employed as an adjuvant to immunotherapy to achieve a more robust immunological response. Some of the most critical limitations of the CRISPR/Cas9 technology include off-target mutations, resulting in nonspecific restrictions of DNA upstream of the Protospacer Adjacent Motifs (PAM), ethical agreements, and the lack of a scientific consensus aiming at risk evaluation. Currently, CRISPR/Cas9 is tested on animal models to enhance genome editing specificity and induce a stronger anti-tumor response. Moreover, ongoing clinical trials use the CRISPR/Cas9 system in immune cells to modify genomes in a target-specific manner. Recently, error-free in vitro systems have been engineered to overcome limitations of this gene-editing system. The aim of the article is to present the knowledge concerning the use of CRISPR Cas9 technique in targeting treatment-resistant cancers. Additionally, the use of CRISPR/Cas9 is aided as an emerging supplementation of immunotherapy, currently used in experimental oncology. Demonstrating further, applications and advances of the CRISPR/Cas9 technique are presented in animal models and human clinical trials. Concluding, an overview of the limitations of the gene-editing tool is proffered.

Dynamic Signatures of the Epigenome: Friend or Foe?

Highly dynamic epigenetic signaling is influenced mainly by (micro)environmental stimuli and genetic factors. The exact mechanisms affecting particular epigenomic patterns differ dependently on the context. In the current review, we focus on the causes and effects of the dynamic signatures of the human epigenome as evaluated with the high-throughput profiling data and single-gene approaches. We will discuss three different aspects of phenotypic outcomes occurring as a consequence of epigenetics interplaying with genotype and environment. The first issue is related to the cases of environmental impacts on epigenetic profile, and its adverse and advantageous effects related to human health and evolutionary adaptation. The next topic will present a model of the interwoven co-evolution of genetic and epigenetic patterns exemplified with transposable elements (TEs) and their epigenetic repressors Krüppel-associated box zinc finger proteins (KRAB-ZNFs). The third aspect concentrates on the mitosis-based microevolution that takes place during carcinogenesis, leading to clonal diversity and expansion of tumor cells. The whole picture of epigenome plasticity and its role in distinct biological processes is still incomplete. However, accumulating data define epigenomic dynamics as an essential co-factor driving adaptation at the cellular and inter-species levels with a benefit or disadvantage to the host.

KRAB ZNF explorer-the online tool for the exploration of the transcriptomic profiles of KRAB-ZNF factors in The Cancer Genome Atlas.

SUMMARY: Krüppel-associated box domain zinc finger protein (KRAB ZNF) explorer is a web-based tool for comprehensive characterization of the mRNA expression status of KRAB-ZNF transcription factors in the data from The Cancer Genome Atlas study. Key functionalities cover a comparative analysis of KRAB-ZNF expression between normal and cancer tissues, an association of KRAB-ZNF expression with various pathological features, including survival analysis, association with global DNA methylation status, analyses of KRAB-ZNF isoform expressions and analysis of KRAB-ZNF levels in normal tissues. AVAILABILITY AND IMPLEMENTATION: KRAB ZNF explorer is available at http://mi2.mini.pw.edu.pl: 8080/KRAB_ZNF/. The source code for shiny application is available at: https://github.com/MI2DataLab/KRAB_ZNF/tree/master/app and the source code for analyses and precalculations are available at: https://github.com/MI2DataLab/KRAB_ZNF/tree/master/work.

The expression signature of cancer-associated KRAB-ZNF factors identified in TCGA pan-cancer transcriptomic data.

The KRAB-ZNF (Krüppel-associated box domain zinc finger) gene family is composed of a large number of highly homologous genes, gene isoforms, and pseudogenes. The proteins encoded by these genes, whose expression is often tissue-specific, act as epigenetic suppressors contributing to the addition of repressive chromatin marks and DNA methylation. Due to its high complexity, the KRAB-ZNF family has not been studied in sufficient detail, and the involvement of its members in carcinogenesis remains mostly unexplored. In this study, we aimed to provide a comprehensive description of cancer-associated KRAB-ZNFs using publicly available The Cancer Genome Atlas pan-cancer datasets. We analyzed 6727 tumor and normal tissue samples from 16 cancer types. Here, we showed that a small but distinctive cluster of 16 KRAB-ZNFs is commonly upregulated across multiple cancer cohorts in comparison to normal samples. We confirmed these observations in the independent panels of lung and breast cancer cell lines and tissues. This upregulation was also observed for most of the KRAB-ZNF splicing variants, whose expression is simultaneously upregulated in tumors compared to normal tissues. Finally, by analyzing the clinicopathological data for breast and lung cancers, we demonstrated that the expression of cancer-associated KRAB-ZNFs correlates with patient survival, tumor histology, and molecular subtyping. Altogether, our study allowed the identification and characterization of KRAB-ZNF factors that may have an essential function in cancer biology and thus potential to become novel oncologic biomarkers and treatment targets.

TRIM28 and Interacting KRAB-ZNFs Control Self-Renewal of Human Pluripotent Stem Cells through Epigenetic Repression of Pro-differentiation Genes.

Reprogramming to induced pluripotent stem cells (iPSCs) and differentiation of pluripotent stem cells (PSCs) are regulated by epigenetic machinery. Tripartite motif protein 28 (TRIM28), a universal mediator of Krüppel-associated box domain zinc fingers (KRAB-ZNFs), is known to regulate both processes; however, the exact mechanism and identity of participating KRAB-ZNF genes remain unknown. Here, using a reporter system, we show that TRIM28/KRAB-ZNFs alter DNA methylation patterns in addition to H3K9me3 to cause stable gene repression during reprogramming. Using several expression datasets, we identified KRAB-ZNFs (ZNF114, ZNF483, ZNF589) in the human genome that maintain pluripotency. Moreover, we identified target genes repressed by these KRAB-ZNFs. Mechanistically, we demonstrated that these KRAB-ZNFs directly alter gene expression of important developmental genes by modulating H3K9me3 and DNA methylation of their promoters. In summary, TRIM28 employs KRAB-ZNFs to evoke epigenetic silencing of its target differentiation genes via H3K9me3 and DNA methylation.

COL1A1, PRPF40A, and UCP2 correlate with hypoxia markers in non-small cell lung cancer.

PURPOSE: Collagen 1A1 (COL1A1), RNA-binding and pre-mRNA Processing Factor (PRPF40A), and Uncoupling Protein 2 (UCP2) were identified as downstream effectors of cytoglobin (CYGB), which was shown implicated in tumour biology. Although these three genes have been previously associated with cancer, little is known about their status in lung malignancies. METHODS: Hereby, we investigated the expression and promoter methylation of COL1A1, PRPF40A, and UCP2 in 156 non-small cell lung cancer (NSCLC) and adjacent normal tissues. RESULTS: We demonstrate that COL1A1 and PRPF40A mRNAs are significantly overexpressed in NSCLC (p < 1 × 10-4), while UCP2 exhibits a trend of upregulation (p = 0.066). Only COL1A1 promoter revealed hypermethylation in NSCLCs (36%), which was particularly evident in squamous cell carcinomas (p = 0.024) and in the tumours with moderate-to-good differentiation (p = 0.01). Transcript level of COL1A1, as well as PRPF40A and UCP2, exhibited striking association (p ≤ 0.001) with the expression of hypoxia markers. In addition, we demonstrate in lung cancer cell lines exposed to hypoxia or oxidative stress that COL1A1 transcription significantly responds to oxygen depletion, while other genes showed the modest upregulation in stress conditions. CONCLUSION: In conclusion, our data revealed that COL1A1, UCP2, and PRPF40A are novel players implicated in the complex network of hypoxia response in NSCLC.

Epigenetic mechanisms of induced pluripotency.

Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) requires profound alterations in the epigenetic landscape. During reprogramming, a change in chromatin structure resets the gene expression and stabilises self-renewal. Reprogramming is a highly inefficient process, in part due to multiple epigenetic barriers. Although many epigenetic factors have already been shown to affect self-renewal and pluripotency in embryonic stem cells (ESCs), only a few of them have been examined in the context of dedifferentiation. In order to improve current protocols of iPSCs generation, it is essential to identify epigenetic drivers and blockages of somatic cell reprogramming.

Computational characterisation of cancer molecular profiles derived using next generation sequencing.

Our current understanding of cancer genetics is grounded on the principle that cancer arises from a clone that has accumulated the requisite somatically acquired genetic aberrations, leading to the malignant transformation. It also results in aberrent of gene and protein expression. Next generation sequencing (NGS) or deep sequencing platforms are being used to create large catalogues of changes in copy numbers, mutations, structural variations, gene fusions, gene expression, and other types of information for cancer patients. However, inferring different types of biological changes from raw reads generated using the sequencing experiments is algorithmically and computationally challenging. In this article, we outline common steps for the quality control and processing of NGS data. We highlight the importance of accurate and application-specific alignment of these reads and the methodological steps and challenges in obtaining different types of information. We comment on the importance of integrating these data and building infrastructure to analyse it. We also provide exhaustive lists of available software to obtain information and point the readers to articles comparing software for deeper insight in specialised areas. We hope that the article will guide readers in choosing the right tools for analysing oncogenomic datasets.

Cytoglobin has bimodal: tumour suppressor and oncogene functions in lung cancer cell lines.

Cytoglobin (CYGB) is frequently downregulated in many types of human malignancies, and its exogenous overexpression reduces proliferation of cancer cells. Despite its implied tumour suppressor (TSG) functions, its exact role in carcinogenesis remains unclear as CYGB upregulation is also associated with tumour hypoxia and aggressiveness. In this study, we explore the TSG role of CYGB, its influence on the phenotype of cancerous cells under stress conditions and the clinical significance of CYGB expression and promoter methylation in non-small cell lung cancer (NSCLC). DNA methylation-dependent expression silencing of CYGB is demonstrated in both clinical samples and cell lines. CYGB promoter was more frequently methylated in lung adenocarcinomas (P = 1.4 × 10(-4)). Demethylation by 5'-azadeoxycytidine partially restored CYGB expression in cell lines. Interestingly, trichostatin A triggered upregulation of CYGB expression in cancer cell lines and downregulation in non-tumourigenic ones. CYGB mRNA expression in NSCLC surgical specimens correlated with that of HIF1α and VEGFa (P < 1 × 10(-4)). Overexpression of CYGB in cancer cell lines reduced cell migration, invasion and anchorage-independent growth. Moreover, CYGB impaired cell proliferation, but only in the lung adenocarcinoma cell line (H358). Upon hydrogen peroxide treatment, CYGB protected cell viability, migratory potential and anchorage independence by attenuating oxidative injury. In hypoxia, CYGB overexpression decreased cell viability, augmented migration and anchorage independence in a cell-type-specific manner. In conclusion, CYGB revealed TSG properties in normoxia but promoted tumourigenic potential of the cells exposed to stress, suggesting a bimodal function in lung tumourigenesis, depending on cell type and microenvironmental conditions.

TPL2 kinase is a suppressor of lung carcinogenesis.

Lung cancer is a heterogeneous disease at both clinical and molecular levels, posing conceptual and practical bottlenecks in defining key pathways affecting its initiation and progression. Molecules with a central role in lung carcinogenesis are likely to be targeted by multiple deregulated pathways and may have prognostic, predictive, and/or therapeutic value. Here, we report that Tumor Progression Locus 2 (TPL2), a kinase implicated in the regulation of innate and adaptive immune responses, fulfils a role as a suppressor of lung carcinogenesis and is subject to diverse genetic and epigenetic aberrations in lung cancer patients. We show that allelic imbalance at the TPL2 locus, up-regulation of microRNA-370, which targets TPL2 transcripts, and activated RAS (rat sarcoma) signaling may result in down-regulation of TPL2 expression. Low TPL2 levels correlate with reduced lung cancer patient survival and accelerated onset and multiplicity of urethane-induced lung tumors in mice. Mechanistically, TPL2 was found to antagonize oncogene-induced cell transformation and survival through a pathway involving p53 downstream of cJun N-terminal kinase (JNK) and be required for optimal p53 response to genotoxic stress. These results identify multiple oncogenic pathways leading to TPL2 deregulation and highlight its major tumor-suppressing function in the lung.

Cytoglobin: biochemical, functional and clinical perspective of the newest member of the globin family.

Since the discovery of cytoglobin (Cygb) a decade ago, growing amounts of data have been gathered to characterise Cygb biochemistry, functioning and implication in human pathologies. Its molecular roles remain under investigation, but nitric oxide dioxygenase and lipid peroxidase activities have been demonstrated. Cygb expression increases in response to various stress conditions including hypoxia, oxidative stress and fibrotic stimulation. When exogenously overexpressed, Cygb revealed cytoprotection against these factors. Cygb was shown to be upregulated in fibrosis and neurodegenerative disorders and downregulated in multiple cancer types. CYGB was also found within the minimal region of a hereditary tylosis with oesophageal cancer syndrome, and its expression was reduced in tylotic samples. Recently, Cygb has been shown to inhibit cancer cell growth in vitro, thus confirming its suggested tumour suppressor role. This article aims to review the biochemical and functional aspects of Cygb, its involvement in various pathological conditions and potential clinical utility.

Neuroglobin and myoglobin in non-small cell lung cancer: expression, regulation and prognosis.

Globins are respiratory proteins involved in oxygen metabolism, which is a critical factor in tumor growth and progression. The status of neuroglobin and myoglobin is largely unknown in human malignancies, including lung cancer. The aim of this study was to explore mRNA expression profiles, potential regulatory mechanisms and clinicopathological associations of neuroglobin and myoglobin in non-small cell lung cancer (NSCLC). We screened 208 surgically resected NSCLC specimens and a panel of lung normal and cancer cell lines. The mRNA expression of neuroglobin, myoglobin and hypoxia markers (HIF1α and VEGFa) was measured with qRTPCR, while neuroglobin promoter methylation was assessed with Pyrosequencing. Neuroglobin and myoglobin were upregulated in the tumor samples compared to normal tissue (p=1.3×10(-22) and p=1.9×10(-9), respectively). Neuroglobin was more frequently overexpressed in squamous cell carcinomas (SqCCL) than adenocarcinomas. Overexpression of myoglobin was more profound in adenocarcinomas, which correlated with poor survival (p=0.013). Neuroglobin promoter was hypermethylated in 30.8% of NSCLC cases, which correlated with neuroglobin mRNA downregulation. The epigenetic regulation of neuroglobin was confirmed by treating lung cell lines with 5'azadeoxycytidine and/or trichostatin A. Expression of both genes correlated with the expression of HIF1α (neuroglobin: p=3.8×10(-5), myoglobin: p=1.1×10(-11)). Myoglobin expression was also associated to that of VEGFa (p=2.1×10(-7)). Hypoxia-dependent upregulation of both globins was validated in vitro. In summary, neuroglobin and myoglobin overexpression in NSCLC is associated with histological subtype, hypoxia and, in case of neuroglobin - epigenetic regulation. Myoglobin expression may have potential significance in the prognostication of lung adenocarcinomas.

UHRF1-mediated tumor suppressor gene inactivation in nonsmall cell lung cancer.

BACKGROUND: The UHRF1 gene possesses an essential role in DNA methylation maintenance, but its contribution to tumor suppressor gene hypermethylation in primary human cancers currently remains unclear. METHODS: mRNA expression levels of UHRF1, DNMT1, DNMT3A, DNMT3B, and E2F1 were evaluated in 105 primary nonsmall cell lung carcinomas by quantitative polymerase chain reaction. The methylation status of CDKN2A and RASSF1 promoters was examined by pyrosequencing. UHRF1 was knocked down by short hairpin RNA in A549 lung adenocarcinoma cells. RESULTS: All 4 genes were overexpressed in a coordinated manner in the lung tumor tissues, and their expression correlated with that of E2F1. Higher UHRF1 expression in tumor tissues correlated with the hypermethylation of CDKN2A (P = .005) and RASSF1 promoters (P = .034), and the relationship with a combined epigenotype was even stronger (P = 2.3 × 10(-4) ). When UHRF1 was knocked down in A549 lung adenocarcinoma cells, lower methylation levels of RASSF1, CYGB, and CDH13 promoters were observed. Also, UHRF1 knockdown clones demonstrated reduced proliferation and decreased cell migration properties. CONCLUSIONS: Our data demonstrate that UHRF1 is a key epigenetic switch, which controls cell cycle in nonsmall cell lung carcinoma through its ability to sustain the transcriptional silencing of tumor suppressor genes by maintaining their promoters in a hypermethylated status. Thus, UHRF1 should be considered, along with DNMTs, among the potential targets for cancer treatment and/or therapeutic stratification.