Abnormal expression of p-ATM/CHK2 in nasal extranodal NK/T cell lymphoma, nasal type, is correlated with poor prognosis.

AIMS: The aim of this study is to investigate the expression profiles of cell cycle related proteins in nasal extranodal NK/T cell lymphoma, nasal type (ENKTCL). METHODS: The expression profiles of cell cycle related proteins were assessed with a cell cycle antibody array and validated by immunohistochemistry. Correlations between the expression levels of proteins and clinical outcomes of patients with nasal ENKTCL were evaluated. RESULTS: The expression of full length ataxia telangiectasia mutated (ATM) in nasal ENKTCL significantly decreased compared with that in nasal benign lymphoid proliferative disease (NBLPD), but the expression levels of p-ATM, CHK2 and RAD51 significantly increased in nasal ENKTCL compared with that in NBLPD. Kaplan-Meier analysis showed that the expression levels of p-ATM and CHK2 in nasal ENKTCL were inversely related to overall survival (p=0.011 and p=0.025, respectively). CONCLUSION: Abnormalities in the ATM pathway may play a crucial role in the oncogenesis and chemoradiotherapy resistance of nasal ENKTCL.

The Expression and Therapeutic Potential of Checkpoint Kinase 2 in Laryngeal Squamous Cell Carcinoma.

INTRODUCTION: Laryngeal squamous cell carcinoma (LSCC) is the most common histological subtype of laryngeal cancer. The involved molecular mechanisms and suitable therapeutic targets for LSCC still need to be further investigated. Checkpoint kinase 2 (CHK2) participates in several cellular physiology pathways and plays a role in tumor progression. However, the roles of CHK2 in LSCC remain unclear. METHODS: mRNA expression data were obtained from The Cancer Genome Atlas (TCGA) database, and bioinformatic analysis was performed. Western blot and immunohistochemical analyses were conducted to detect protein expression. MTS assays were performed to examine cell growth of LSCC-derived cell lines. RESULTS: In the present study, we found that both active form of CHK2 and total CHK2 protein expressions were up-regulated in LSCC tissues. Positive expression of CHK2 was closely associated with advanced clinical features and poor prognosis. Moreover, potential CHK2-involving bioprocesses and signaling pathways were analyzed. In addition, repressed proliferation of LSCC cells was induced by CHK2 inhibitor. DISCUSSION: Taken together, our findings elucidated that CHK2 may act as an oncogenic factor in LSCC, suggesting a potential target for clinical treatment.

Effects of Cancer Genetic Panel Testing on at-Risk Individuals.

OBJECTIVE: To evaluate the role of screening patients at increased risk for hereditary cancer syndromes with an extended panel of cancer predisposition genes to identify actionable genetic mutations. METHODS: A retrospective chart review was conducted of all patients presenting to a multidisciplinary cancer program for genetic counseling and testing from January 2015 to December 2016. Individuals presenting to the program were identified as at-risk by a personal or family history of cancer, by their health care provider, or by self-referral. All participants met current National Comprehensive Cancer Network criteria for genetic risk evaluation for hereditary cancer. The results of testing and its implications for management, based on National Comprehensive Cancer Network guidelines, were recorded. RESULTS: Of 670 at-risk patients who underwent genetic testing, 66 (9.9%) had BRCA-limited testing; of these, 26 of 670 (3.9%) had a deleterious or likely pathogenic mutation. Expanded panel testing was done for 560 of the 670 patients (83.4%), and abnormal results were found in 65 of 670 (9.7%); non-BRCA mutations (predominantly CHEK2) were found in 49 of the 65 (75%). Abnormal genetic testing was associated with increased surveillance in 96% of those with deleterious mutations, whereas negative testing for a known familial mutation in 45 patients was associated with a downgrade of their risk and reduction of subsequent surveillance and management. CONCLUSION: Guideline-based management is frequently altered by genetic testing, including panel testing, in patients at risk for cancer. We recommend that obstetrics and gynecology providers routinely refer at-risk patients for genetic counseling and testing when clinically appropriate.

CHK2 stability is regulated by the E3 ubiquitin ligase SIAH2.

The serine threonine checkpoint kinase 2 (CHK2) is a critical protein involved in the DNA damage-response pathway, which is activated by phosphorylation inducing cellular response such as DNA repair, cell-cycle regulation or apoptosis. Although CHK2 activation mechanisms have been amply described, very little is known about degradation control processes. In the present study, we identify the ubiquitin E3 ligase SIAH2 as an interaction partner of CHK2, which mediates its ubiquitination and proteasomal degradation. CHK2 degradation is independent of both its activation and its kinase activity, but also of the phosphorylation in S456. We show that SIAH2-deficient cells present CHK2 accumulation together with lower ubiquitination levels. Accordingly, SIAH2 depletion by siRNA increases CHK2 levels. In response to DNA damage induced by etoposide, interaction between both proteins is disrupted, thus avoiding CHK2 degradation and promoting its stabilization. We also found that CHK2 phosphorylates SIAH2 at three residues (Thr26, Ser28 and Thr119), modifying its ability to regulate certain substrates. Cellular arrest in the G2/M phase induced by DNA damage is reverted by SIAH2 expression through the control of CHK2 levels. We observed that hypoxia decreases CHK2 levels in parallel to SIAH2 induction. Similarly, we provide evidence suggesting that resistance to apoptosis induced by genotoxic agents in cells subjected to hypoxia could be partly explained by the mutual regulation between both proteins. These results indicate that SIAH2 regulates CHK2 basal turnover, with important consequences on cell-cycle control and on the ability of hypoxia to alter the DNA damage-response pathway in cancer cells.

Analyzing DNA replication checkpoint in budding yeast.

Checkpoints are conserved mechanisms that prevent progression into the next phase of the cell cycle when cells are unable to accomplish the previous event properly. Cells also possess a surveillance mechanism called the DNA replication checkpoint, which consists of a conserved kinase cascade that is provoked by insults that block or slow down replication fork progression. In the budding yeast Saccharomyces cerevisiae, the DNA replication checkpoint controls the timing of S-phase events such as origin firing and spindle elongation. This checkpoint also upregulates dNTP pools and maintains the replication fork structure in order to resume DNA replication after replication block. Many replication checkpoint factors have been found to be tumor suppressors, highlighting the importance of this checkpoint pathway in human health. Here we describe a series of protocols to analyze the DNA replication checkpoint in S. cerevisiae.

DNA damage response-related proteins in gastric cancer: ATM, Chk2 and p53 expression and their prognostic value.

OBJECTIVES: The aims of this study were to assess expressions of the DNA damage response (DDR)-related proteins and to investigate their clinical significances in gastric carcinoma. METHODS: Two independent cohorts, a training set (n=524) and validation set (n=394), of gastric cancer patients were enrolled. Ataxia telangiectasia mutated (ATM), checkpoint kinase 2 (Chk2), and p53 expressions were examined by immunohistochemistry using tissue microarray. RESULTS: ATM loss, Chk2 loss, and p53 positivity were observed in 21.8, 14.1, and 36.1% of the training set, and in 17.3, 12.2, and 35.8% of the validation set, respectively. In the training set, the aberrant expressions of ATM, Chk2, or p53 were significantly associated with an advanced TNM stage and poor disease-specific survival. This association was verified in the validation set. Chk2 positivity and p53 negativity were significantly related to a prolonged disease-specific survival. Also, patients with nonaberrant expressional levels of all 3 DDR-related proteins had a more favorable outcome than others. Multivariate analyses showed that Chk2 loss and at least 1 aberrant DDR-related protein remained as independent prognostic factors of poor disease-specific survival. CONCLUSIONS: This study elucidated the prognostic implications of DDR-related proteins, and suggests that their aberrant expressions play critical roles in the development and progression of gastric cancer.