Overexpression Of UHRF1 Research Articles

Role of UHRF1 in methylation regulation and angiogenesis

Many recent studies have proved that ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is an important nuclear protein associated with tumorigenesis, which plays a significant role in epigenetic regulation, especially in DNA methylation and histone methylation. For its particular domains, UHRF1 plays a critical role in biological behaviors including cell proliferation, cell cycle, and apoptosis. Overexpression of UHRF1 in various tumors is closely associated with the angiogenesis in tumors. This paper will provide a review of the regulation of UHRF1 in DNA methylation and histone methylation, and discuss the potential epigenetic role of UHRF1 in angiogenesis.

MiR-506 Targets UHRF1 to Inhibit Colorectal Cancer Proliferation and Invasion via the KISS1/PI3K/NF-κB Signaling Axis.

BackgroundThe UHRF1 gene is an epigenetic modification factor that mediates tumor suppressor gene silencing in a variety of cancers. Related studies have reported that UHRF1 can inhibit the expression of the KISS1 gene. However, the regulatory mechanism underlying UHRF1 expression in colorectal cancer (CRC) is still unclear. The aim of this study was to gain a better understanding of the regulation of UHRF1 expression in CRC and to determine whether it regulates the mechanism by which KISS1 promotes CRC metastasis.MethodsIn the present study, the levels of miR-506, UHRF1 and KISS1 expression in CRC tissues and in human CRC cell lines were studied using quantitative real-time PCR (qRT-PCR) and Western blotting. Cell proliferation, migration, and invasion assays are used to detect cell proliferation, migration, and invasion. A dual-luciferase reporter system was used to confirm the target gene of miR-506.ResultsThis study found that UHRF1 protein is highly expressed in CRC tissues and negatively correlated with KISS1 protein expression. UHRF1 overexpression activates the PI3K/NF-κB signaling pathway by inhibiting the mRNA expression levels of pathway mediators. Bioinformatics analysis and luciferase reporter gene assays confirmed that miR-506 targets UHRF1.ConclusionThis study identified the regulation of UHRF1 expression in CRC and the mechanism of CRC metastasis. UHRF1 may be a new potential target molecule for future CRC metastasis treatment.

Overexpression of UHRF1 and its potential role in the development of invasive ductal breast cancer validated by integrative bioinformatics and immunohistochemistry analyses.

BackgroundIncreasing evidence has highlighted the role of ubiquitin-like PHD and RING finger domain-containing protein 1 (UHRF1) in the development of cancers, including hepatocellular carcinoma, pancreatic cancer, and bladder cancer. However, the correlation between UHRF1 and breast cancer remains unclear. The present study aimed to analyze the expression of UHRF1 and its role in the development of invasive ductal breast cancer (IDC) by integrating multilevel expression data and immunohistochemistry analysis.MethodsThe Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases were used to gather UHRF1 expression data on IDC. Additionally, immunohistochemistry analysis was used to investigate the correlations between UHRF1 expression and the clinical characteristics of IDC.ResultsThe GEO and TCGA databases indicated that UHRF1 was up-regulated in IDC. Consistently, the immunohistochemical specimens showed that the significant overexpression of UHRF1 in IDC, and its expression level showed an increasing trend from ductal carcinomas in situ to IDC. Notably, the increased levels of UHRF1 were closely correlated with estrogen receptor expression, pathological grade, and the prognosis of the disease. In addition, patients with a high UHRF1 expression had a poorer prognosis.ConclusionsIn conclusion, our findings suggested that UHRF1 plays a promoting role in breast tumorigenesis, and the over-expression of UHRF1 could serve as a biomarker for the prognosis in invasive ductal carcinomas in breast cancer.

UHRF1 Promotes Proliferation of Human Adipose-Derived Stem Cells and Suppresses Adipogenesis via Inhibiting Peroxisome Proliferator-Activated Receptor γ.

Once the adipose tissue is enlarged for the purpose of saving excess energy intake, obesity may be observed. Ubiquitin-like with PHD and RING Finger domains 1 (UHRF1) is helpful in repairing damaged DNA as it increases the resistance of cancer cells against cytocidal drugs. Peroxisome proliferator-activated receptor γ (PPARγ), an important nucleus transcription factor participating in adipogenesis, has been extensively reported. To date, no study has indicated whether UHRF1 can regulate proliferation and differentiation of human adipose-derived stem cells (hADSCs). Hence, this study aimed to utilize overexpression or downregulation of UHRF1 to explore the possible mechanism of proliferation and differentiation of hADSCs. We here used lentivirus, containing UHRF1 (LV-UHRF1) and siRNA-UHRF1 to transfect hADSCs, on which Cell Counting Kit-8 (CCK-8), cell growth curve, colony formation assay, and EdU proliferation assay were applied to evaluate proliferation of hADSCs, cells cycle was investigated by flow cytometry, and adipogenesis was detected by Oil Red O staining and Western blotting. Our results showed that UHRF1 can promote proliferation of hADSCs after overexpression of UHRF1, while proliferation of hADSCs was reduced through downregulation of UHRF1, and UHRF1 can control proliferation of hADSCs through transition from G1-phase to S-phase; besides, we found that UHRF1 negatively regulates adipogenesis of hADSCs via PPARγ. In summary, the results may provide a new insight regarding the role of UHRF1 on regulating proliferation and differentiation of hADSCs.

Abstract 2148: UHRF1 overexpression is a significant driver of tumor progression in RB-mediated cancers

Abstract UHRF1 is an essential epigenetic reader and writer overexpressed in multiple human cancers, including retinoblastoma and osteosarcoma. For these tumors, metastasis remains the most significant fatal complication for treatment. Genetic alterations at the RB transcriptional corepressor 1 (RB1) gene are associated with initiation, increased mortality, metastasis, and poor response to chemotherapy. However, the precise mechanism(s) through which this occurs remains to be elucidated. Studies in our laboratory identified UHRF1 (Ubiquitin-like, containing PHD and RING Finger domains 1) as a gene that is upregulated and its protein overexpressed in retinoblastoma and osteosarcoma. UHRF1 is a multifunctional protein involved in epigenetic regulation and has been shown to have protein-protein interactions with RB. Further, the RB/E2F pathway directly regulates UHRF1 expression. Using genetic engineered mouse models, we found that UHRF1 is essential for retinoblastoma formation. We have also studied the role of UHRF1 in other RB-dependent malignancies, including osteosarcoma. Our results indicate that UHRF1 is overexpressed in most human osteosarcomas. Loss-of-function studies using UHRF1 shRNA knockdowns and CRISPR/Cas9 knockouts indicate that reduction of UHRF1 levels in human osteosarcoma cells results in decreased proliferation, migration and invasion. Strikingly, induction of UHRF1 overexpression in normal mesenchymal stem cells induces increased cell migration and invasion. We evaluated the role of UHRF1 in osteosarcoma development by knocking-out Uhrf1 in genetically engineered mouse models of osteosarcoma (p53 cKO: Osx-cre p53lox/lox and p53/Rb1 DKO: Osx-cre p53lox/lox Rb1lox/lox), to create p53/Uhrf1 DKO (Osx-cre p53lox/lox Uhrf1lox/lox) and p53/Rb1/Uhrf1 TKO (Osx-cre p53lox/lox Rb1lox/lox Uhrf1lox/lox) mice. Uhrf1 abrogation leads to a significant increase in survival in p53/Rb1/Uhrf1 TKO mice compared to their p53/Rb1 DKO littermate controls. While 100% of p53/Rb1 DKO mice develop tumors, 12.5% of p53/Rb1/Uhrf1 TKO mice did not, confirmed using microCT/PET imaging. Further, p53/Rb1/Uhrf1 TKO showed a significant reduction in the rate and number of lung metastasic nodules compared to p53/Rb1 DKO. Even though UHRF1 is overexpressed in osteosarcomas with wild type Rb1 (p53 cKO tumors), we found no significant difference in the median survival of p53/Uhrf1 DKO compared to p53 cKO littermate controls. These results suggest that UHRF1 overexpression is a significant contributor of the poor prognosis associated with loss of Rb1. Altogether, our data indicates that UHRF1 overexpression plays a significant role in osteosarcoma proliferation, migration, invasion, and metastasis, which translates in reduced survival mice with osteosarcoma. Our studies suggest that UHRF1 may play a role in carcinogenesis and is a putative biomarker and anticancer therapeutic target. Citation Format: Claudia A. Benavente, Stephanie C. Wu, Loredana Zocchi. UHRF1 overexpression is a significant driver of tumor progression in RB-mediated cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2148.

TET Upregulation Leads to 5-Hydroxymethylation Enrichment in Hepatoblastoma.

Hepatoblastoma is an embryonal liver tumor carrying few genetic alterations. We previously disclosed in hepatoblastomas a genome-wide methylation dysfunction, characterized by hypermethylation at specific CpG islands, in addition to a low-level hypomethylation pattern in non-repetitive intergenic sequences, in comparison to non-tumoral liver tissues, shedding light into a crucial role for epigenetic dysregulation in this type of cancer. To explore the underlying mechanisms possibly related to aberrant epigenetic modifications, we evaluated the expression profile of a set of genes engaged in the epigenetic machinery related to DNA methylation (DNMT1, DNMT3A, DNMT3B, DNMT3L, UHRF1, TET1, TET2, and TET3), as well as the 5-hydroxymethylcytosine (5hmC) global level. We observed in hepatoblastomas a general disrupted expression of these genes from the epigenetic machinery, mainly UHRF1, TET1, and TET2 upregulation, in association with an enrichment of 5hmC content. Our findings support a model of active demethylation by TETs in hepatoblastoma, probably during early stages of liver development, which in combination with UHRF1 overexpression would lead to DNA hypomethylation and an increase in overall 5hmC content. Furthermore, our data suggest that decreased 5hmC content might be associated with poor survival rate, highlighting a pivotal role of epigenetics in hepatoblastoma development and progression.

UHRF1 promotes renal cell carcinoma progression through epigenetic regulation of TXNIP.

UHRF1 is an important epigenetic regulator that belongs to the UHRF family. Overexpression of UHRF1 has been found in many kinds of tumors and its overexpression is associated with poor prognosis and short survival in certain cancer types. However, its function in renal cell carcinoma (RCC) is not clear. Here we report that RCC tumor tissues had obviously higher UHRF1 expression than normal renal tissues. Downregulation of UHRF1 by siRNA or shRNA in RCC cell lines resulted in decreased cell viability, inhibited cell migration and invasion, and increased apoptosis. UHRF1 knockdown RCC xenografts also resulted in obviously inhibited tumor growth in vivo. After downregulation of UHRF1 in RCC cells, the expression of TXNIP was upregulated. In addition, after UHRF1 and TXNIP were simultaneously downregulated, cell viability and cell invasion increased, whereas cell apoptosis decreased compared with UHRF1 single downregulated cells. We also showed that UHRF1 could recruit HDAC1 to the TXNIP promoter and mediate the deacetylation of histone H3K9, resulting in the inhibition of TXNIP expression. Our results confirm that UHRF1 has oncogenic function in RCC and UHRF1 may promote tumor progression through epigenetic regulation of TXNIP. UHRF1 might be used as a therapeutic target for RCC treatment.

UHRF genes regulate programmed interdigital tissue regression and chondrogenesis in the embryonic limb

The primordium of the limb contains a number of progenitors far superior to those necessary to form the skeletal components of this appendage. During the course of development, precursors that do not follow the skeletogenic program are removed by cell senescence and apoptosis. The formation of the digits provides the most representative example of embryonic remodeling via cell degeneration. In the hand/foot regions of the embryonic vertebrate limb (autopod), the interdigital tissue and the zones of interphalangeal joint formation undergo massive degeneration that accounts for jointed and free digit morphology. Developmental senescence and caspase-dependent apoptosis are considered responsible for these remodeling processes. Our study uncovers a new upstream level of regulation of remodeling by the epigenetic regulators Uhrf1 and Uhrf2 genes. These genes are spatially and temporally expressed in the pre-apoptotic regions. UHRF1 and UHRF2 showed a nuclear localization associated with foci of methylated cytosine. Interestingly, nuclear labeling increased in cells progressing through the stages of degeneration prior to TUNEL positivity. Functional analysis in cultured limb skeletal progenitors via the overexpression of either UHRF1 or UHRF2 inhibited chondrogenesis and induced cell senescence and apoptosis accompanied with changes in global and regional DNA methylation. Uhrfs modulated canonical cell differentiation factors, such as Sox9 and Scleraxis, promoted apoptosis via up-regulation of Bak1, and induced cell senescence, by arresting progenitors at the S phase and upregulating the expression of p21. Expression of Uhrf genes in vivo was positively modulated by FGF signaling. In the micromass culture assay Uhrf1 was down-regulated as the progenitors lost stemness and differentiated into cartilage. Together, our findings emphasize the importance of tuning the balance between cell differentiation and cell stemness as a central step in the initiation of the so-called “embryonic programmed cell death” and suggest that the structural organization of the chromatin, via epigenetic modifications, may be a precocious and critical factor in these regulatory events.

Overexpression of UHRF1 promoted the proliferation of vascular smooth cells via the regulation of Geminin protein levels.

Geminin is an inhibitor of DNA replication licensing and cell cycle. Our previous study demonstrates that Geminin plays an important role in regulating phenotypic diversity and growth of vascular smooth cells (VSMCs). Ubiquitin-like with PHD and RING Finger domains 1 (UHRF1) is an epigenetic coordinator, whose RING domain confers intrinsic E3 ligase activity, mediating the ubiquitination of several proteins and the protein–protein interaction. Aberrant expression of UHRF1 was related to aggressiveness of multiple human malignancies, where knockdown of UHRF1 led to decreased proliferation of cancer cells. However, it is unclear whether proper UHRF1 function is involved in aberrant proliferation and phenotypic switching of VSMCs via altering Geminin protein levels. In present study, in UHRF1-overexpressing A10 cells, 3H-thymidine and 5-ethynyl-20-deoxyuridine (EdU) and CCK8 were used to examine the proliferation of VSMCs. RT-PCR and Western blot analyses were performed to investigate whether UHRF1-mediated effects were achieved by altering Geminin expression in VSMCs. RNA-seq analysis was performed to dissect related mechanisms or signaling pathways of these effects. The results of in vitro experiments suggested that UHRF1 prompted proliferation and cell cycle of VSMCs via the down-regulation of Geminin protein levels with no change in Geminin mRNA expression. Besides, PI3K-Akt signaling pathway was increased upon UHRF1 up-regulation. Our study demonstrated that overexpressing UHRF1 was involved in VSMCs proliferation through reducing inhibitory Geminin protein levels to promote cell cycle as well as activating PI3K-Akt signaling. This may provide key knowledge for the development of better strategies to prevent diseases related to VSMCs abnormal proliferation.

Activated MEK/ERK Pathway Drives Widespread and Coordinated Overexpression of UHRF1 and DNMT1 in Cancer cells

The UHRF1-DNMT1 axis plays a key role in DNA maintenance methylation in mammals. Accumulative studies demonstrate that UHRF1 is broadly overexpressed in cancers, which contributes to cancer cell proliferation and tumorigenesis. Interestingly, a proteasome-dependent downregulation of UHRF1 has been observed in pluripotent ground state mouse embryonic stem cells (mESCs) cultured in the presence of two kinase (MEK1/MEK2 and GSK3β) inhibitors (termed 2i), raising the question whether UHRF1 is similarly regulated in cancer cells. Here we present evidence that while addition of 2i broadly downregulates UHRF1 and DNMT1 in various cancer cells, distinct underlying mechanisms are involved. In contrast to mESCs, 2i-induced downregulation of UHRF1 and DNMT1 in cancer cells cannot be rescued by proteasome inhibitor and occurs primarily at the level of transcription. Furthermore, downregulation of UHRF1 and DNMT1 by 2i is due to inhibition of MEK1/MEK2, but not GSK3β activity. Data mining reveals a marked co-expression of UHRF1 and DNMT1 in normal tissues as well as cancers. We provide evidence that multiple transcription factors including E2F1 and SP1 mediate the transcriptional activation of UHRF1 and DNMT1 by the activated MEK/ERK pathway. Together our study reveals distinct regulation of UHRF1/DNMT1 in mESCs and cancer cells and identifies activated MEK/ERK pathway as a driving force for coordinated and aberrant over-expression of UHRF1 and DNMT1 in cancers.

Epigenetic mechanism and target therapy of UHRF1 protein complex in malignancies.

Ubiquitin-like with plant homeodomain and really interesting new gene finger domains 1 (UHRF1) functions as an epigenetic regulator recruiting PCNA, DNMT1, histone deacetylase 1, G9a, SuV39H, herpes virus-associated ubiquitin-specific protease, and Tat-interactive protein by multiple corresponding domains of DNA and H3 to maintain DNA methylation and histone modifications. Overexpression of UHRF1 has been found as a potential biomarker in various cancers resulting in either DNA hypermethylation or global DNA hypo-methylation, which participates in the occurrence, progression, and invasion of cancer. The role of UHRF1 in the reciprocal interaction between DNA methylation and histone modifications, the dynamic structural transformation of UHRF1 protein within epigenetic code replication machinery in epigenetic regulations, as well as modifications during cell cycle and chemotherapy targeting UHRF1 are evaluated in this study.

Abstract A41: Role of UHRF1 in tumorigenesis and metastasis of osteosarcoma

Abstract Osteosarcoma is the eighth most common form of childhood cancer, with very limited advances in treatment over the past three decades. Osteosarcoma is a highly metastatic cancer, with 15-20% of osteosarcoma patients diagnosed after the cancer has already metastasized, causing the survival rate to be little over 50%. Common sites of metastasis include lung and liver. Loss of the retinoblastoma protein (RB) is associated with poor prognosis in osteosarcoma, as defined by shorter life span and heavier tumor burden. Ubiquitin Like with PHD and Ring Finger Domains 1 (UHRF1) has been identified as a critical downstream effector of the RB/E2F pathway that is overexpressed in various cancers. We have confirmed using human mesenchymal stem cells (MSCs) and osteosarcoma cell lines that UHRF1 is directly regulated by the RB/E2F pathway in the bone lineage, and found that the UHRF1 gene is upregulated and its protein overexpressed in osteosarcoma. Using gain/loss-of-function assays to study the role of UHRF1 in osteosarcoma, we observed that UHRF1 is involved in promoting cell migration. To study the role of UHRF1 in osteosarcoma tumor development, we generated Uhrf1 conditional knockout mice. In this system, we use an Osterix-driven Cre recombinase (Osx-cre) to facilitate preosteoblast-specific loss of various combinations of genes. Osx-cre Tp53; Rb1 double knockout mice give rise to osteosarcoma and have a median survival of 26.7 weeks. Additional loss of Uhrf1 in Osx-cre Tp53; Rb1 double knockout mice (Osx-cre Tp53; Rb1; Uhrf1 triple knockout mice) significantly delays the age of tumor onset to >47 weeks. Furthermore, triple knockout mice have a decreased frequency and number of lung and liver metastasis. In conclusion, we have strong evidences both in vitro and in vivo that UHRF1 is a crucial component in aiding osteosarcoma progression, and have identified its novel role in tumor metastasis. The high metastatic rate and low 5-year survival rate following metastasis of osteosarcoma make UHRF1 a promising target for therapeutics. Further studies on the mechanism by which UHRF1 overexpression contributes to osteosarcomagenesis, including tumor growth and dissemination, will strongly aid the process of drug development for anticancer treatment. Citation Format: Stephanie C. Wu, Claudia A. Benavente. Role of UHRF1 in tumorigenesis and metastasis of osteosarcoma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A41.

Overexpression of UHRF1 promotes silencing of tumor suppressor genes and predicts outcome in hepatoblastoma

BackgroundHepatoblastoma (HB) is the most common liver tumor of childhood and occurs predominantly within the first 3 years of life. In accordance to its early manifestation, HB has been described to display an extremely low mutation rate. As substitute, epigenetic modifiers seem to play an exceptional role in its tumorigenesis, which holds promise to develop targeted therapies and establish biomarkers for patient risk stratification.ResultsWe examined the role of a newly described protein complex consisting of three epigenetic regulators, namely E3 ubiquitin-like containing PHD and RING finger domain 1 (UHRF1), ubiquitin-specific-processing protease 7 (USP7), and DNA methyltransferase 1 (DNMT1), in HB. We found the complex to be located on the promoter regions of the pivotal HB-associated tumor suppressor genes (TSGs) HHIP, IGFBP3, and SFRP1 in HB cells, thereby leading to strong repression through DNA methylation and histone modifications. Consequently, knockdown of UHRF1 led to DNA demethylation and loss of the repressive H3K9me2 histone mark at the TSG loci with their subsequent transcriptional reactivation. The observed growth impairment of HB cells upon UHRF1 knockdown could be attributed to reduced expression of genes involved in cell cycle progression, negative regulation of cell death, LIN28B signaling, and the adverse 16-gene signature, as revealed by global RNA sequencing. Clinically, overexpression of UHRF1 in primary tumor tissues was significantly associated with poor survival and the prognostic high-risk 16-gene signature.ConclusionThese findings suggest that UHRF1 is critical for aberrant TSG silencing and sustained growth signaling in HB and that UHRF1 overexpression levels might serve as a prognostic biomarker and potential molecular target for HB patients.

Targeting microRNA/UHRF1 pathways as a novel strategy for cancer therapy.

Ubiquitin-like containing plant homeodomain and RING finger domains 1 (UHRF1) is an anti-apoptotic protein involved in the silencing of several tumor suppressor genes (TSGs) through epigenetic modifications including DNA methylation and histone post-translational alterations, and also epigenetic-independent mechanisms. UHRF1 overexpression is observed in a number of solid tumors and hematological malignancies, and is considered a primary mechanism in inhibiting apoptosis. UHRF1 exerts its inhibitory activity on TSGs by binding to functional domains and therefore influences several epigenetic actors including DNA methyltransferase, histone deacetylase 1, histone acetyltransferase Tat-interacting protein 60 and histone methyltransferases G9a and Suv39H1. UHRF1 is considered to control a large macromolecular protein complex termed epigenetic code replication machinery, in order to maintain epigenetic silencing of TSGs during cell division, thus enabling cancer cells to escape apoptosis. MicroRNAs (miRNAs) are able to regulate the expression of its target gene by functioning as either an oncogene or a tumor suppressor. In the present review, the role of tumor suppressive miRNAs in the regulation of UHRF1, and the importance of targeting the microRNA/UHRF1 pathways in order to induce the reactivation of silenced TSGs and subsequent apoptosis are discussed.

Globular adiponectin inhibits leptin-stimulated esophageal adenocarcinoma cell proliferation via adiponectin receptor 2-mediated suppression of UHRF1.

Esophageal adenocarcinoma (EAC) is one of the most common malignancies in the world which is associated the increased prevalence of obesity. In the context of obesity, leptin can directly contribute to progression of EAC. Adiponectin inhibits leptin-induced oncogenic signaling in EAC cells. However, the exact molecular mechanisms linking obesity, adipokines, and EAC remain far from completely understood. In the present study, we tested the role of ubiquitin-like with PHD and ring finger domains 1 (UHRF1) in adiponectin-induced protective effects against leptin-induced EAC cell proliferation. We found that globular adiponectin (gAD) significantly inhibited leptin-induced increase of cell proliferation and decrease of apoptosis in OE 19 cells. Moreover, leptin-induced increase of UHRF1 expression was suppressed by gAD. Compared with normal controls, UHRF1 expression was markedly increased in EAC tissues and cell lines. Silence of UHRF1 increased the expression of cleaved caspase 3 and 9 and Bax, reduced the expression of Bcl-2, promoted apoptosis, and inhibited cell proliferation in OE 19 cells. Overexpression of UHRF1 significantly blocked gAD-induced decrease of cell proliferation and increase of apoptosis in leptin-treated cells. Silence of adiponectin receptor 1/2 (AdipoR1/2) could inhibit gAD-induced decrease of cell proliferation and increase of apoptosis in leptin-treated cells. Silence of AdipoR2, but not AdipoR1, suppressed gAD-induced decrease of UHRF1 expression in leptin-treated cells. The results indicated that gAD inhibited the prooncogenic effects of leptin via AdipoR2-mediated suppression of UHRF1. Our study provides novel insights into the role of UHRF1 in the development of EAC and the mechanism of antitumor effect of gAD.

The Ubiquitin-like with PHD and Ring Finger Domains 1 (UHRF1)/DNA Methyltransferase 1 (DNMT1) Axis Is a Primary Regulator of Cell Senescence

As senescence develops, cells sequentially acquire diverse senescent phenotypes along with simultaneous multistage gene reprogramming. It remains unclear what acts as the key regulator of the collective changes in gene expression at initiation of senescent reprogramming. Here we analyzed time series gene expression profiles obtained in two different senescence models in human diploid fibroblasts: replicative senescence and H2O2-induced senescence. Our results demonstrate that suppression of DNA methyltransferase 1 (DNMT1)-mediated DNA methylation activity was an initial event prior to the display of senescent phenotypes. We identified seven DNMT1-interacting proteins, ubiquitin-like with PHD and ring finger domains 1 (UHRF1), EZH2, CHEK1, SUV39H1, CBX5, PARP1, and HELLS (also known as LSH (lymphoid-specific helicase) 1), as being commonly down-regulated at the same time point as DNMT1 in both senescence models. Knockdown experiments revealed that, among the DNMT1-interacting proteins, only UHRF1 knockdown suppressed DNMT1 transcription. However, UHRF1 overexpression alone did not induce DNMT1 expression, indicating that UHRF1 was essential but not sufficient for DNMT1 transcription. Although UHRF1 knockdown effectively induced senescence, this was significantly attenuated by DNMT1 overexpression, clearly implicating the UHRF1/DNMT1 axis in senescence. Bioinformatics analysis further identified WNT5A as a downstream effector of UHRF1/DNMT1-mediated senescence. Senescence-associated hypomethylation was found at base pairs -1569 to -1363 from the transcription start site of the WNT5A gene in senescent human diploid fibroblasts. As expected, WNT5A overexpression induced senescent phenotypes. Overall, our results indicate that decreased UHRF1 expression is a key initial event in the suppression of DNMT1-mediated DNA methylation and in the consequent induction of senescence via increasing WNT5A expression.

Abstract P1-08-11: UHRF1 promotes breast cancer progression via suppressing KLF17 expression

Abstract Background: UHRF1, also termed as ICBP90 or Np95, is reported to be an epigenetic regulator of DNA methylation and histone modifications, which takes pivotal functions in cell differentiation and tumorigenesis. Here, we show that UHRF1 is markedly increased in breast cancer tissues and cell lines. And patients with high UHRF1 expression have a poor prognosis compare with those in low UHRF1 expression as showed by Kaplan Meier plotter analysis. However, the underlying mechanisms of UHRF1 in breast cancer remain largely unknown. Thus, the aim of this study is to validate potential mechanisms of UHRF1 in breast cancer cells. Methods: The stable cell lines were constructed using interference and overexpression methods and the efficiency was confirmed by western blot. Then cell proliferation and migration were evaluated by CCK8 and transwell assay. The MDA-MB-231 pCDH and pCDH-UHRF1 cells were inoculate into mouse left flank to further testify UHRF1 function in vivo. Furthermore, microarray and ChIP-seq were implemented to detect potential mechanisms and the candidate genes were assessed by ChIP and qPCR. The BSP or MSP primers were designed by the methprimer database. Genomic DNA was prepared from breast cancer cells and then the genomic DNA was bisulfite modified using EZ DNA Methylation-Gold Kit. The methylation PCR products were cloned into pMD-18T and ten positive clones were sequenced. The data were analyzed using the QUMA analyzer software. Results: We found that UHRF1 was indeed increased in breast cancer tissues and cells. And patients with high levels of UHRF1 were likely to have a shorter disease-free survival than patients with low levels. UHRF1 overexpression can promote cell proliferation and migration while UHRF1 downregulation have inverse functions. In vivo, UHRF1 also accelerate tumor growth on mice. Mechanistic studies revealed that 9 candidate genes were obtained by analysis from ChIP-seq and microarray databases. The further verification showed that Krüppel-like factor 17 (KLF17) is the most significantly upregulated one when UHRF1 gene was depleted, with rich CpG islands on its promoter region. We also observed that an inverse relationship between the expression of UHRF1 and KLF17 both in breast cancer cell lines and tissues.What's more, cells proliferation and migration which were inhibited by UHRF1-depletion can be rescued by KLF17 silence, suggesting UHRF1 promote breast cancer proliferation and migration through downregulating KLF17 expression. Moreover, overexpression of UHRF1 increases the methylation of CpG nucleotides and reduces the expression of KLF17 while depletion of UHRF1 decreases the methylation of CpG nucleotides with elevating expression of KLF17. Conclusion: Our results demonstrate that increased UHRF1 can promote breast cancer cell proliferation and migration by epigenetic silencing of KLF17 expression through CpG islands methylation in its promoter region. These results provide insight into the breast cancer progression process and suggest that making changes in this mechanism represent new therapeutic approaches to block breast cancer development. Citation Format: Gao S-P, Sun H-F, Li L-D, Jin W. UHRF1 promotes breast cancer progression via suppressing KLF17 expression [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-08-11.

UHRF1: The key regulator of epigenetics and molecular target for cancer therapeutics.

UHRF1 is a master regulator of epigenome as it coordinates DNA methylation and histone modifications. Compelling evidence suggests a strong link between UHRF1 overexpression and tumorigenesis, substantiating its ability to act as a potential biomarker for cancer diagnosis and prognosis. UHRF1 also mediates repair of damaged DNA that makes cancer cells resistant toward cytocidal drugs. Hence, understanding the molecular mechanism of UHRF1 regulation would help in developing cancer therapeutics. Natural compounds have shown applicability to downregulate UHRF1 leading to growth arrest and apoptosis in cancer cells.

UHRF1 is an Independent Prognostic Factor and a Potential Therapeutic Target of Esophageal Squamous Cell Carcinoma.

Purpose: Ubiquitin-like with plant homeodomain and ring-finger domains 1 (UHRF1) plays an essential role in DNA methylation, and the overexpression of UHRF1 is associated with poor prognosis in various cancers. Esophageal squamous cell carcinoma (ESCC) accounts for approximately 90% of esophageal cancer cases in China, but the five-year survival rate for patients is less than 10% due to limited clinical approaches for early diagnosis and treatment. The present research aimed to investigate the expression of UHRF1 in ESCC and its biological role in ESCC development.Methods: UHRF1 expression in ESCC and normal esophageal tissues was examined using immunohistochemical staining, followed by analysis of the correlation between UHRF1 expression and clinical features. In addition, the effects of lentivirus-mediated RNA interference of UHRF1 on global DNA methylation, cell proliferation, cell cycle progression and apoptosis and were investigated in ESCC cells.Results: UHRF1 was overexpressed in ESCC tissues and was an independent prognostic factor for ESCC patients. In ESCC cells, knockdown of UHRF1 caused global DNA hypomethylation, inhibited cell proliferation and induced apoptosis. Furthermore, UHRF1 depletion induced cell cycle arrest at the G2/M phase, accompanied by activation of Wee1 and DNA damage response pathway.Conclusions: Our findings identify UHRF1 as a promising prognostic marker for ESCC and suggest that UHRF1 may be a potential therapy target for ESCC patients with elevated UHRF1 expression.

UHRF1 regulates global DNA hypomethylation and is associated with poor prognosis in esophageal squamous cell carcinoma.

BackgroundGlobal DNA hypomethylation contributes to oncogenesis through various mechanisms. The level of long interspersed nucleotide element-1 (LINE- 1) methylation is considered a surrogate marker of global DNA methylation, and is attracting interest as a good predictor of cancer prognosis. However, the mechanism how LINE-1 (global DNA) methylation is controlled in cancer cells remains to be fully elucidated. Ubiquitin-like with PHD and RING finger domain 1 (UHRF1) plays a crucial role in DNA methylation. UHRF1 is overexpressed in many cancers, and UHRF1 overexpression may be a mechanism underlying DNA hypomethylation in cancer cells. Nonetheless, the relationship between UHRF1, LINE-1 methylation level, and clinical outcome in esophageal squamous cell carcinoma (ESCC) remains unclear.ResultsIn ESCC cell lines, vector-mediated UHRF1 overexpression caused global DNA (LINE-1) hypomethylation and, conversely, UHRF1 knockdown using siRNA increased the global DNA methylation level. In ESCC tissues, UHRF1 expression was significantly associated with LINE-1 methylation levels. Furthermore, UHRF1 overexpression correlated with poor prognosis in our cohort of 160 ESCC patients.Materials and MethodsThe relationships between UHRF1 expression and LINE-1 methylation level (i.e., global DNA methylation level) were investigated using ESCC tissues and cell lines. In addition, we examined the correlation between UHRF1 expression, LINE-1 methylation, and clinical outcome in patients with ESCC.ConclusionsOur results suggest that UHRF1 is a key epigenetic regulator of DNA methylation and might be a potential target for cancer treatment.