Phosphorylation-Dependent Regulation of the DNA Damage Response of Adaptor Protein KIBRA in Cancer Cells.
01 May 2016-Molecular and Cellular Biology (American Society for Microbiology)-Vol. 36, Iss: 9, pp 1354-1365
TL;DR: The imperative functional role of KIBRA per se is demonstrated (KIBRA phosphorylation at T1006 site as a molecular switch that regulates the DNA damage response, possibly via the nonhomologous end joining [NHEJ] pathway), suggesting that K IBRA could be a potential therapeutic target for modulating chemoresistance in cancer cells.
Abstract: Multifunctional adaptor proteins encompassing various protein-protein interaction domains play a central role in the DNA damage response pathway. In this report, we show that KIBRA is a physiologically interacting reversible substrate of ataxia telangiectasia mutated (ATM) kinase. We identified the site of phosphorylation in KIBRA as threonine 1006, which is embedded within the serine/threonine (S/T) Q consensus motif, by site-directed mutagenesis, and we further confirmed the same with a phospho-(S/T) Q motif-specific antibody. Results from DNA repair functional assays such as the γ-H2AX assay, pulsed-field gel electrophoresis (PFGE), Comet assay, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and clonogenic cell survival assay using stable overexpression clones of wild-type (wt.) KIBRA and active (T1006E) and inactive (T1006A) KIBRA phosphorylation mutants showed that T1006 phosphorylation on KIBRA is essential for optimal DNA double-strand break repair in cancer cells. Further, results from stable retroviral short hairpin RNA-mediated knockdown (KD) clones of KIBRA and KIBRA knockout (KO) model cells generated by a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system showed that depleting KIBRA levels compromised the DNA repair functions in cancer cells upon inducing DNA damage. All these phenotypic events were reversed upon reconstitution of KIBRA into cells lacking KIBRA knock-in (KI) model cells. All these results point to the fact that phosphorylated KIBRA might be functioning as a scaffolding protein/adaptor protein facilitating the platform for further recruitment of other DNA damage response factors. In summary, these data demonstrate the imperative functional role of KIBRAper se(KIBRA phosphorylation at T1006 site as a molecular switch that regulates the DNA damage response, possibly via the nonhomologous end joining [NHEJ] pathway), suggesting that KIBRA could be a potential therapeutic target for modulating chemoresistance in cancer cells.
Citations
More filters
TL;DR: The present review aims at giving an overview about the evolution of this widely applicable method from the first description by Ostling and Johanson to the OECD Guideline 489 for the in vivo mammalian comet assay.
Abstract: The comet assay is a versatile, reliable, cost-efficient, and fast technique for detecting DNA damage and repair in any tissue. It is useable in almost any cell type and applicable to both eukaryotic and prokaryotic organisms. Instead of highlighting one of the numerous specific aspects of the comet assay, the present review aims at giving an overview about the evolution of this widely applicable method from the first description by Ostling and Johanson to the OECD Guideline 489 for the in vivo mammalian comet assay. In addition, methodical aspects and the influence of critical steps of the assay as well as the evaluation of results and improvements of the method are reviewed. Methodical aspects regarding oxidative DNA damage and repair are also addressed. An overview about the most recent works and relevant cutting-edge reviews based on the comet assay with special regard to, e.g., clinical applications, nanoparticles or environmental risk assessment concludes this review. Taken together, the presented overview raises expectations to further decades of successful applications and enhancements of this excellent method.
100 citations
TL;DR: A novel exosome-based mechanism that may underlie a cancer cell's ability to survive radiation is identified that is inhibited induction of proliferation and cellular survival.
Abstract: Radiation is utilized in the therapy of more than 50% of cancer patients. Unfortunately, many malignancies become resistant to radiation over time. We investigated the hypothesis that one method of a cancer cell's ability to survive radiation occurs through cellular communication via exosomes. Exosomes are cell-derived vesicles containing DNA, RNA, and protein. Three properties were analyzed: 1) exosome function, 2) exosome profile and 3) exosome uptake/blockade. To analyze exosome function, we show radiation-derived exosomes increased proliferation and enabled recipient cancer cells to survive radiation in vitro. Furthermore, radiation-derived exosomes increased tumor burden and decreased survival in an in vivo model. To address the mechanism underlying the alterations by exosomes in recipient cells, we obtained a profile of radiation-derived exosomes that showed expression changes favoring a resistant/proliferative profile. Radiation-derived exosomes contain elevated oncogenic miR-889, oncogenic mRNAs, and proteins of the proteasome pathway, Notch, Jak-STAT, and cell cycle pathways. Radiation-derived exosomes contain decreased levels of tumor-suppressive miR-516, miR-365, and multiple tumor-suppressive mRNAs. Ingenuity pathway analysis revealed the most represented networks included cell cycle, growth/survival. Upregulation of DNM2 correlated with increased exosome uptake. To analyze the property of exosome blockade, heparin and simvastatin were used to inhibit uptake of exosomes in recipient cells resulting in inhibited induction of proliferation and cellular survival. Because these agents have shown some success as cancer therapies, our data suggest their mechanism of action could be limiting exosome communication between cells. The results of our study identify a novel exosome-based mechanism that may underlie a cancer cell's ability to survive radiation.
48 citations
TL;DR: Analysis of data retrieved from patients who had one of six cancer subtypes as well as healthy persons indicated that these genes could be important biomarkers for discriminating different cancer sub types and healthy controls.
Abstract: // Yu-Hang Zhang 1, 2, * , Tao Huang 2, * , Lei Chen 4, * , YaoChen Xu 5 , Yu Hu 2 , Lan-Dian Hu 2 , Yudong Cai 3 and Xiangyin Kong 2 1 Department of General Surgery, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, People’s Republic of China 2 Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, People’s Republic of China 3 School of Life Sciences, Shanghai University, Shanghai 200444, People’s Republic of China 4 College of Information Engineering, Shanghai Maritime University, Shanghai 201306, People’s Republic of China 5 Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, People’s Republic of China * These authors have contributed equally to this work Correspondence to: Lan-Dian Hu, email: ldhu2013@163.com Yudong Cai, email: cai_yud@126.com Xiangyin Kong, email: xykong@sibs.ac.cn Keywords: cancer detection, liquid biopsy, RNA-seq data, support vector machine, maximum relevance minimum redundancy Received: June 15, 2017 Accepted: August 16, 2017 Published: September 15, 2017 ABSTRACT Detection and diagnosis of cancer are especially important for early prevention and effective treatments. Traditional methods of cancer detection are usually time-consuming and expensive. Liquid biopsy, a newly proposed noninvasive detection approach, can promote the accuracy and decrease the cost of detection according to a personalized expression profile. However, few studies have been performed to analyze this type of data, which can promote more effective methods for detection of different cancer subtypes. In this study, we applied some reliable machine learning algorithms to analyze data retrieved from patients who had one of six cancer subtypes (breast cancer, colorectal cancer, glioblastoma, hepatobiliary cancer, lung cancer and pancreatic cancer) as well as healthy persons. Quantitative gene expression profiles were used to encode each sample. Then, they were analyzed by the maximum relevance minimum redundancy method. Two feature lists were obtained in which genes were ranked rigorously. The incremental feature selection method was applied to the mRMR feature list to extract the optimal feature subset, which can be used in the support vector machine algorithm to determine the best performance for the detection of cancer subtypes and healthy controls. The ten-fold cross-validation for the constructed optimal classification model yielded an overall accuracy of 0.751. On the other hand, we extracted the top eighteen features (genes), including TTN, RHOH, RPS20, TRBC2, in another feature list, the MaxRel feature list, and performed a detailed analysis of them. The results indicated that these genes could be important biomarkers for discriminating different cancer subtypes and healthy controls.
47 citations
Cites background from "Phosphorylation-Dependent Regulatio..."
...Among our candidate subtypes, this gene has been confirmed to contribute to all six cancer subtypes, implying that this gene may be a functional marker to distinguish healthy samples from specific cancer subtypes [50-55]....
[...]
TL;DR: The current and emerging regulatory roles of KIBRA and PTPN14 in the Hippo pathway and their functions in cancer are summarized.
Abstract: The Hippo signaling pathway regulates cellular proliferation and survival, thus exerting profound effects on normal cell fate and tumorigenesis Pivotal effectors of this pathway are YAP/TAZ, transcriptional co-activators whose dysfunction contributes to the development of cancer Complex networks of intracellular and extracellular signaling pathways that modulate YAP and TAZ activities have recently been identified Among them, KIBRA and PTPN14 are two evolutionarily-conserved and important YAP/TAZ upstream regulators They can negatively regulate YAP/TAZ functions separately or in concert In this review, we summarize the current and emerging regulatory roles of KIBRA and PTPN14 in the Hippo pathway and their functions in cancer
24 citations
Cites background from "Phosphorylation-Dependent Regulatio..."
...Very recently, KIBRA has been found to interact with ataxia telangiectasia mutated (ATM), whose phosphorylation at T1006 is necessary for optimal DNA double-strand break repair in cancer cells [39]....
[...]
TL;DR: Overall low-KIBRA expression has an independent effect on the RFS and predicts the Rfs outcome of luminal breast cancer patients who received endocrine therapy and breast cancer Patients who received chemotherapy.
Abstract: This study was carried out to evaluate the prognostic value of KIBRA in breast cancer. This retrospective study included breast cancer patients who sought the services of the immunohistochemistry laboratory of our unit from 2006 to 2015. Tissue microarrays were constructed and immunohistochemical staining was done to assess the KIBRA expression. The Kaplan-Meier model for univariate and Cox-regression model with backward stepwise factor retention method for multivariate analyses were used. Chi square test was used to find out the associations with the established prognostic features. A total of 1124 patients were included in the study and KIBRA staining of 909 breast cancers were available for analysis. Cytoplasmic KIBRA expression was seen in 39.5% and nuclear expression in 44.8%. Overall KIBRA–low breast cancers accounted for 41.5%. KIBRA nuclear expression was significantly associated with positive ER and PR expression. Luminal breast cancer patients who had endocrine therapy and KIBRA-low expression had a RFS disadvantage over those who were positive for KIBRA (p = 0.02). Similarly, patients who received chemotherapy and had overall KIBRA-low expression also demonstrated a RFS disadvantage compared to those who had overall positive KIBRA expression (p = 0.018). This effect of KIBRA was independent of the other factors considered for the model. Overall low-KIBRA expression has an independent effect on the RFS and predicts the RFS outcome of luminal breast cancer patients who received endocrine therapy and breast cancer patients who received chemotherapy.
8 citations
Cites background from "Phosphorylation-Dependent Regulatio..."
...Further investigations revealed that KIBRA could be a potential therapeutic target for modulating chemo-resistance in cancer cells [4]....
[...]
...DNA repair function of KIBRA has been demonstrated to modulate chemoresistance in cancer cells in KIBRA knockout and knock-in model cells [4]....
[...]
References
More filters
TL;DR: A set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies are described.
Abstract: Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.
8,663 citations
TL;DR: In this article, a method for producing high-titer, helper-free infectious retroviruses is disclosed which employs a novel strategy that uses transient transfection of new retroviral producer cell lines, ecotropic line BOSC 23 and amphotropic line CAK 8.
Abstract: A method for producing high-titer, helper-free infectious retroviruses is disclosed which employs a novel strategy that uses transient transfection of new retroviral producer cell lines, ecotropic line BOSC 23 and amphotropic line CAK 8, both of which cell lines and their precursor cell lines are disclosed. Because of the advantages over stable packaging cell lines, the BOSC 23 and CAK 8 transient transfection systems greatly facilitate and extend the use of helper-free retroviral vectors. The cell lines and corresponding methods possess wide application in both the medical and biotechnical fields, including gene therapy. These potential applications are disclosed and illustrated.
2,587 citations
TL;DR: Understanding ATM's mode of action provides new insights into the association between defective responses to DNA damage and cancer, and brings us closer to resolving the issue of cancer predisposition in some A-T carriers.
Abstract: Maintenance of genome stability is essential for avoiding the passage to neoplasia. The DNA-damage response--a cornerstone of genome stability--occurs by a swift transduction of the DNA-damage signal to many cellular pathways. A prime example is the cellular response to DNA double-strand breaks, which activate the ATM protein kinase that, in turn, modulates numerous signalling pathways. ATM mutations lead to the cancer-predisposing genetic disorder ataxia-telangiectasia (A-T). Understanding ATM's mode of action provides new insights into the association between defective responses to DNA damage and cancer, and brings us closer to resolving the issue of cancer predisposition in some A-T carriers.
2,579 citations
"Phosphorylation-Dependent Regulatio..." refers background in this paper
...Upon sensing DNA damage signal, ATM becomes activated by its phosphorylation at serine 1981 (19)....
[...]
TL;DR: The evidence presented strongly supports a role for the gamma-H2AX and the PI-3 protein kinase family in focus formation at sites of double-strand breaks and suggests the possibility of a change in chromatin structure accompanying double-Strand break repair.
2,107 citations
"Phosphorylation-Dependent Regulatio..." refers background in this paper
...The loss of -H2AX at DSB sites is thought to reflect the completion of DNA repair at break sites (20, 21)....
[...]
TL;DR: The results clearly establish ATM as the major kinase involved in the phosphorylation of H2AX and suggest that ATM is one of the earliest kinases to be activated in the cellular response to double-strand breaks.
1,909 citations