Zhigang Guo

OGG1 is essential in oxidative stress induced DNA demethylation.

DNA demethylation is an essential cellular activity to regulate gene expression; however, the mechanism that triggers DNA demethylation remains unknown. Furthermore, DNA demethylation was recently demonstrated to be induced by oxidative stress without a clear molecular mechanism. In this manuscript, we demonstrated that 8-oxoguanine DNA glycosylase-1 (OGG1) is the essential protein involved in oxidative stress-induced DNA demethylation. Oxidative stress induced the formation of 8-oxoguanine (8-oxoG). We found that OGG1, the 8-oxoG binding protein, promotes DNA demethylation by interacting and recruiting TET1 to the 8-oxoG lesion. Downregulation of OGG1 makes cells resistant to oxidative stress-induced DNA demethylation, while over-expression of OGG1 renders cells susceptible to DNA demethylation by oxidative stress. These data not only illustrate the importance of base excision repair (BER) in DNA demethylation but also reveal how the DNA demethylation signal is transferred to downstream DNA demethylation enzymes.

Targeting DNA Flap Endonuclease 1 to Impede Breast Cancer Progression

DNA flap endonuclease 1 (FEN1) plays critical roles in maintaining genome stability and integrity by participating in both DNA replication and repair. Suppression of FEN1 in cells leads to the retardation of DNA replication and accumulation of unrepaired DNA intermediates, resulting in DNA double strand breaks (DSBs) and apoptosis. Therefore, targeting FEN1 could serve as a potent strategy for cancer therapy. In this study, we demonstrated that FEN1 is overexpressed in breast cancers and is essential for rapid proliferation of cancer cells. We showed that manipulating FEN1 levels in cells alters the response of cancer cells to chemotherapeutic drugs. Furthermore, we identified a small molecular compound, SC13 that specifically inhibits FEN1 activity, thereby interfering with DNA replication and repair in vitro and in cells. SC13 suppresses cancer cell proliferation and induces chromosome instability and cytotoxicity in cells. Importantly, SC13 sensitizes cancer cells to DNA damage-inducing therapeutic modalities and impedes cancer progression in a mouse model. These findings could establish a paradigm for the treatment of breast cancer and other cancers as well.

Feedback inhibition of CREB signaling by p38 MAPK contributes to the negative regulation of steroidogenesis

BackgroundSteroidogenesis is a complex, multi-steps biological process in which, cholesterol precursor is converted to steroids in a tissue specific and tropic hormone dependent manner. Given that steroidogenesis is achieved by coordinated functioning of multiple tissue specific enzymes, many steroids intermediates/metabolites are generated during this process. Both the steroid products as well as major lipoprotein cholesterol donor, high-density lipoprotein 3 (hHDL3) have the potential to negatively regulate steroidogenesis via increased oxidative stress/reactive oxygen species (ROS) generation.MethodsIn the current study, we examined the effects of treatment of a mouse model of steroidogenesis, Y1-BS1 adrenocortical tumor cells with pregnenolone, 22(R)-Hydroxycholesterol [22(R)-diol] or hHDL3 on ROS production, phosphorylation status of p38 MAPK and cAMP response element-binding protein (CREB), CREB transcriptional activity and mRNA expression of StAR, CPY11A1/P450scc and antioxidant enzymes, superoxide dismutases [Cu,ZnSOD (SOD1), MnSOD (SOD2)], catalase (CAT) and glutathione peroxidase 1 (GPX1). We also detected the steroid product in p38 MAPK inhibitor treated Y1 cells by HPLC-MS / MS.ResultsTreatment of Y1 cells with H2O2 greatly enhanced the phosphorylation of both p38 MAPK and CREB protein. Likewise, treatment of cells with pregnenolone, 22(R) diol or hHDL3 increased ROS production measured with the oxidation-sensitive fluorescent probe 2′,7′-Dichlorofluorescin diacetate (DCFH-DA). Under identical experimental conditions, treatment of cells with these agents also increased the phosphorylation of p38 MAPK and CREB. This increased CREB phosphorylation however, was associated with its decreased transcriptional activity. The stimulatory effects of pregnenolone, 22(R)-diol and hHDL3 on CREB phosphorylation was abolished by a specific p38 MAPK inhibitor, SB203580. Pregnenolone, and 22(R) diol but not hHDL3 upregulated the mRNA expression of SOD1, SOD2 and GPX1, while down-regulated the mRNA levels of StAR and CYP11A1. The p38 inhibitor SB203580 could increase the steroid production in HDL3, 22(R)-diol or pregnenolone treated cells.ConclusionOur data demonstrate induction of a ROS/p38 MAPK -mediated feedback inhibitory pathway by oxy-cholesterol and steroid intermediates and products attenuates steroidogenesis via inhibition of CREB transcriptional activity.

FEN1 promotes tumor progression and confers cisplatin resistance in non‐small‐cell lung cancer

Lung cancer is one of the leading causes of cancer mortality worldwide. The therapeutic effect of chemotherapy is limited due to the resistance of cancer cells, which remains a challenge in cancer therapeutics. In this work, we found that flap endonuclease 1 (FEN1) is overexpressed in lung cancer cells. FEN1 is a major component of the base excision repair pathway for DNA repair systems and plays important roles in maintaining genomic stability through DNA replication and repair. We showed that FEN1 is critical for the rapid proliferation of lung cancer cells. Suppression of FEN1 resulted in decreased DNA replication and accumulation of DNA damage, which subsequently induced apoptosis. Manipulating the amount of FEN1 altered the response of lung cancer cells to chemotherapeutic drugs. A small-molecule inhibitor (C20) was used to target FEN1 and this enhanced the therapeutic effect of cisplatin. The FEN1 inhibitor significantly suppressed cell proliferation and induced DNA damage in lung cancer cells. In mouse models, the FEN1 inhibitor sensitized lung cancer cells to a DNA damage-inducing agent and efficiently suppressed cancer progression in combination with cisplatin treatment. Our study suggests that targeting FEN1 may be a novel and efficient strategy for a tumor-targeting therapy for lung cancer.

Telomerase antagonist imetelstat increases radiation sensitivity in esophageal squamous cell carcinoma

The morbidity and mortality of esophageal cancer is one of the highest around the world and the principal therapeutic method is radiation. Thus, searching for sensitizers with lower toxicity and higher efficiency to improve the efficacy of radiation therapy is critical essential. Our research group has previously reported that imetelstat, the thio-phosphoramidate oligonucleotide inhibitor of telomerase, can decrease cell proliferation and colony formation ability as well as increase DNA breaks induced by radiation in esophageal cancer cells. Further study in this project showed that imetelstat significantly sensitized esophageal cancer cells to radiation in vitro. Later study showed that imetelstat leads to increased cell apoptosis. We also measured the expression level of several DNA repair and apoptosis signaling proteins. pS345 CHK1, γ-H2AX, p53 and caspase3 expression were up-regulated in imetelstat treated cells, identifying these factors as molecular markers. Mouse in vivo model using imetelstat at clinically achievable concentrations and fractionated irradiation scheme yielded results demonstrating radiosensitization effect. Finally, TUNEL assay, caspase 3 and Ki67 staining in tumor tissue proved that imetelstat sensitized esophageal cancer to radiation in vivo through promoting cell apoptosis and inhibiting cell proliferation. Our study supported imetelstat increase radiation sensitivity of esophageal squamous cell carcinoma through inducing cell apoptosis and the specific inhibitor of telomerase might serve as a potential novel therapeutic tool for esophageal squamous cell carcinoma therapy.

Mutation of DNA Polymerase β R137Q Results in Retarded Embryo Development Due to Impaired DNA Base Excision Repair in Mice

DNA polymerase β (Pol β), a key enzyme in the DNA base excision repair (BER) pathway, is pivotal in maintaining the integrity and stability of genomes. One Pol β mutation that has been identified in tumors, R137Q (arginine to glutamine substitution), has been shown to lower polymerase activity, and impair its DNA repair capacity. However, the exact functional deficiency associated with this polymorphism in living organisms is still unknown. Here, we constructed Pol β R137Q knock-in mice, and found that homozygous knock-in mouse embryos were typically small in size and had a high mortality rate (21%). These embryonic abnormalities were caused by slow cell proliferation and increased apoptosis. In R137Q knock-in mouse embryos, the BER efficiency was severely impaired, which subsequently resulted in double-strand breaks (DSBs) and chromosomal aberrations. Furthermore, R137Q mouse embryo fibroblasts (MEFs) were more sensitive to DNA-damaging reagents, such as methyl methanesulfonate (MMS) and H2O2. They displayed a higher percentage of DSBs, and were more likely to undergo apoptosis. Our results indicate that R137 is a key amino acid site that is essential for proper Pol β functioning in maintaining genomic stability and embryo development.

NHERF1 and NHERF2 regulation of SR-B1 stability via ubiquitination and proteasome degradation

Scavenger receptor class B type 1 (SR-B1), an HDL receptor plays a crucial role in cholesterol metabolism in the liver, steroidogenic tissues, and vascular cells including macrophages. SR-B1 is subject to regulation at the transcription, posttranscription and posttranslational levels. We previously provided evidence that PDZ domain containing NHERF1 and NHERF2 regulate SR-B1 protein levels post-transcriptionally, although the underlying mechanism(s) by which NHERF1 and NHERF2 regulate SR-B1 protein levels is not well understood. In this study, we demonstrate that SR-B1 is degraded intracellularly via ubiquitin-proteasome pathway and that SR-B1 can be ubiquitinated at K500 and K508 residues. Overexpression of NHERF1 or NHERF2 enhanced SR-B1 ubiquitination and degradation. NHERF1 and NHERF2 promote SR-B1 ubiquitination at sites K508 and K500, respectively. These results suggest that NHERF1 and NHERF2 down-regulated SR-B1 at least in part via the ubiquitin/proteasome pathway.

Prominent role of histone lysine demethylases in cancer epigenetics and therapy

Protein methylation has an important role in the regulation of chromatin, gene expression and regulation. The protein methyl transferases are genetically altered in various human cancers. The enzymes that remove histone methylation have led to increased awareness of protein interactions as potential drug targets. Specifically, Lysine Specific Demethylases (LSD) removes methylated histone H3 lysine 4 (H3K4) and H3 lysine 9 (H3K9) through formaldehyde-generating oxidation. It has been reported that LSD1 and its downstream targets are involved in tumor-cell growth and metastasis. Functional studies of LSD1 indicate that it regulates activation and inhibition of gene transcription in the nucleus. Here we made a discussion about the summary of histone lysine demethylase and their functions in various human cancers.

Wnt pathway is involved in 5-FU drug resistance of colorectal cancer cells.

Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. 5-Fluorouracil (5-FU) is widely used in the treatment of cancers, but its antineoplastic activity is limited in drug-resistant cancer cells. To investigate the detailed mechanism of 5-FU resistance, we developed a model of 5-FU-resistant cells from HCT-8 cells, a well-established colorectal cancer cell line. We found that the drug-resistant cells demonstrated high expression of TCF4 and β-catenin, indicating an upregulated Wnt pathway. A microarray analysis revealed that the suppression of the checkpoint kinase 1 (CHK1) pathway explained the resistance to 5-FU, especially in p53 wild-type cancer cells such as HCT-8. Our data also demonstrated that the CHK1 pathway is suppressed by the Wnt pathway in 5-FU-resistant cells. In summary, we have discovered a novel mechanism for 5-FU resistance mediated by histone deacetylation, which also revealed the crosstalk between the Wnt pathway and CHK1 pathway.Colorectal cancer: pathways to drug resistanceThe interaction between two signaling pathways may be related to resistance to a leading cancer drug in colorectal cancer cells. The drug 5-fluorouracil (5-FU) is widely used to limit malignant cell proliferation, but some cancers, including colorectal cancers, are resistant to 5-FU. Zhigang Guo at Nanjing Normal University, China, and co-workers have uncovered the signaling pathways behind 5-FU resistance in colorectal cancer cell lines. They found that 5-FU-resistant cells expressed high levels of two proteins indicative of an over-active Wnt signaling pathway, a crucial pathway in healthy development, the disruption of which is implicated in many diseases. This upregulated pathway suppressed another signaling pathway, leading to reduced cancer cell death. This interaction between the pathways could prove to be a valuable therapeutic target to limit resistance to 5-FU.

Synergistic antitumor effect of combined paclitaxel with FEN1 inhibitor in cervical cancer cells

Studies on cervical cancer are urgently required to improve clinical outcomes. As a major anticancer drug for cervical cancer, paclitaxel has been used for many years in clinical therapy but its therapeutic efficacy is limited by common obstacle from cancer cells. The enhanced DNA repair pathways of cancer cells have been proved to survive DNA damage induced by chemotherapeutic drug. Inhibitors of specific DNA repair pathway can sensitize cancer cells to the treatment of chemotherapeutic drugs. In this paper we found that the effect of paclitaxel can be significantly improved when used in combination with FEN1 inhibitor SC13, suggesting a synergistic mechanism between the two compounds. Our studies suggest that FEN1 inhibition could be a novel strategy of tumor-targeting therapy for cervical cancer. Our work also revealed that paclitaxel demonstrates stronger synergistic effect with SC13 than other common used chemical drugs such as doxorubicin, carboplatin or camptothecin on cervical cancer cells.