CHK2 Mutations Research Articles

Computational analysis of high-risk SNPs in human CHK2 gene responsible for hereditary breast cancer: A functional and structural impact.

Nowadays CHK2 mutation is studied frequently in hereditary breast and ovarian cancer patients in addition to BRCA1/BRCA2. CHK2 is a tumor suppressor gene that encodes a serine/threonine kinase, also involved in pathways such as DNA repair, cell cycle regulation and apoptosis in response to DNA damage. CHK2 is a well-studied moderate penetrance gene that correlates with third high risk susceptibility gene with an increased risk for breast cancer. Hence before planning large population study, it is better to scrutinize putative functional SNPs of CHK2 using different computational tools. In this study, we have used various computational approaches to identify nsSNPs which are deleterious to the structure and/or function of CHK2 protein that might be causing this disease. Computational analysis was performed by different in silico tools including SIFT, Align GVGD, SNAP-2, PROVEAN, Poly-Phen-2, PANTHER, PhD-SNP, MUpro, iPTREE-STAB, Consurf, InterPro, NCBI Conserved Domain Search tool, ModPred, SPARKS-X, RAMPAGE, Verify-3D, FT Site, COACH and PyMol. Out of 78 nsSNP of human CHK2 gene, seven nsSNPs were predicted functionally most significant SNPs. Among these seven nsSNP, p.Arg160Gly, p.Gly210Arg and p.Ser415Phe are highly conserved residues with conservation score of 9 and three nsSNP were predicted to be involved in post translational modification. The p.Arg160Gly and p.Gly210Arg may interfere in phosphopeptide binding site on FHA conserved domain. The p.Ser415Phe may interfere in formation of activation loop of protein-kinase domain and might interfere in interactions of CHK2 with ligand. The study concludes that mutation of serine to phenylalanine at position 415 is a major mutation in native CHK2 protein which might contribute to its malfunction, ultimately causing disease. This is the first comprehensive study, where CHK2 gene variants are analyzed using in silico tools hence it will be of great help while considering large scale studies and also in developing precision medicines related to these polymorphisms in the era of personalized medicine.

Abstract 3815: CHEK2 mutation is an adverse prognostic survival factor for patients diagnosed with high-grade serous ovarian carcinoma

Abstract High-grade serous ovarian carcinoma (HG-SOC) is a poorly prognosed and lethal gynecological disease where only 30% of patients survive beyond five years after initial pathologic diagnosis. However, the heterogeneity of HG-SOC tumors meant that patients often respond differently despite similar clinical intervention. Therefore, identification of novel molecular tumor subtypes/classes, tumor-driving mutations and prognostically significant patient subgroups could provide improvement in clinical management as well as facilitate our understanding of this complex disease and subsequent design of targeted personalized therapeutics. In this work, we performed integrative bioinformatics analyses of mutational and clinical data of HG-SOC patients from The Cancer Genome Atlas(TCGA) and aim to identify the driver genes and their mutations with emphasis on their prognostic significance and molecular functions which could be used as molecular targets for personalization of HG-SOC therapeutics. We identified a 27-gene mutagenesis signature from the study of 334 HG-SOC patients, and our results indicated that the use of this signature can significantly stratify a subgroup of 24 patients with very poor overall survival prognosis (p-value=3.8E-09); all these 24 patients did not survive beyond five years after initial diagnosis. Among these 27 genes, physiological functions such as kinase activity or ATP binding were enriched, and suggest that targeting of these specific cellular programs could be employed to modify tumors' response to therapy and patients' overall survival time. Mutations in CHEK2, RPS6KA2 and MLL4 strongly co-occurred in tumor cells of the poor prognosis patient subgroup which in turn, were under-represented with mutations of BRCA1 or BRCA2. CHEK2 mutations were significantly correlated with poor patient prognosis (p-value=0.00012) and poor response to therapy (p-value =0.046). In 22 of the 24 HG-SOC patients, CHEK2 mutations occurred within a nuclear localization signal (NLS). Results from computational modeling, coupled with literature support, suggest that mutations within the NLS locus could potentially inhibit nuclear import of the Chk2 protein and lead to reduction of Chk2 protein levels in the nucleus. Our findings revealed a previously uncharacterized genetic subtype of HG-SOCs hallmarked with the 27 mutated genes which could result in the functional aberrations in interactions and pathways associated with the genes, possibly leading to very poor prognosis and chemotherapeutic response. We identified a cluster of mutations in the final exon of CHEK2 that selectively predict poor prognosis allowing us to stratify patients that might benefit from therapeutic strategies that target aberrant Chk2 oncogenic signaling in HG-SOC progression. Experiments that will validate the impact of Chk2 mutations are currently underway. Citation Format: Ghim Siong Ow, Anna V. Ivshina, Gloria Fuentes, Vladimir A. Kuznetsov. CHEK2 mutation is an adverse prognostic survival factor for patients diagnosed with high-grade serous ovarian carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3815. doi:10.1158/1538-7445.AM2014-3815

In Vivo Radiobiological Characterization of Proton Beam at the National Cancer Center in Korea: Effect of the Chk2 Mutation

The relative biological effectiveness (RBE) in the presence or absence of CHK2 was estimated at the Korean National Cancer Center Proton Therapy Center (NCCPTC). The proton beam was fixed at 210 MeV with 6-cm spread-out Bragg peaks (SOBPs) because this is expected to be the most frequently used clinical setting. X-rays were obtained using a 6-MV conventional linear accelerator. The RBE was estimated from the survival of jejunal crypt in C3H/He and Chk2(-/-) mice. The estimated RBEs of the NCCPTC at the middle of the SOBP were 1.10 and 1.05 in the presence and absence of CHK2, respectively. The doses that reduced the number of regenerated crypt per jejunal circumference to 20 (D(20)) in C3H/He mice were 14.8 Gy (95% confidence interval [CI], 13.7-15.9) for X-rays and 13.5 Gy (95% CI, 14.5-15.5) for protons. By contrast, the doses of D(20) in Chk2(-/-) mice were 15.7 Gy (95% CI, 15.0-16.4) and 14.9 Gy (95% CI, 14.0-15.8) for X-rays and protons, respectively. The RBE of the NCCPTC is clearly within the range of RBEs determined at other facilities and is consistent with the generic RBE value of 1.10 for 150- to 250-MeV beams. The mutation of Chk2 gave rise to radioresistance but exhibited similar RBE.

PTEN Promoter Silencing and Cowden Syndrome

THE PHOSPHATASE AND TENSIN HOMOLOG GENE (PTEN) (RefSeq NM_000314) is a tumor suppressor located on the human chromosome 10q arm and is an important mediator of carcinogenesis in a variety of human malignancies. By the strictest definition, a tumor suppressor is a gene whose loss confers an increased lifetime risk of developing tumors. The most illustrative examples of genes that fulfill this criterion are those associated with familial cancer syndromes whereby heritable inactivation of 1 allele and subsequent increased tumor risk is passed along to each generation in an autosomaldominant fashion. Using this as a framework, PTEN is a bona fide tumor suppressor gene in that heritable germline mutations have been described in Cowden syndrome (CS), giving rise to a number of human tumors and cancers, most notably thyroid and breast cancers. As is the paradigm of tumor suppressor genes, affected patients with CS inherit 1 mutant inactive copy of PTEN from either parent, and the ensuing loss of the second allele results in tumor formation with subsequent genetic events that eventually lead to cancer. Although there are notable exceptions to this model, most heritable cancer syndromes are believed to adhere to this pattern. Although anomalies within a specific gene often account for the majority of a given familial cancer syndrome, there are striking examples in which a pedigree matches the syndrome, yet no mutations are found in the affected individuals. Such perplexing cases have led to the identification of additional genes that can account for a significant fraction in these cases. For example, germline mutations of the TP53 gene account for the majority of families with LiFraumeni syndrome, a heritable cancer syndrome hallmarked by a number of different cancers, including breast cancers, sarcomas, and leukemias. However, in families diagnosed with Li-Fraumeni syndrome without a TP53 mutation, germline mutations in the cell cycle checkpoint gene CHK2 can account for a considerable proportion of these familial cases. Similarly, patients with CS generally have been found to have germline mutations in PTEN, but in a substantial number of these families no PTEN mutations have been found. Along the same lines of CHK2 mutations and Li-Fraumeni syndrome, germline mutations in the succinate dehydrogenase subunits SDHB-D have been previously described in patients with PTEN wild-type CS as well as individuals who have features of CS but do not meet diagnostic criteria: patients who are said to have Cowdenlike syndrome (CLS). Despite the discovery of germline mutations in these genes, there remains a sizeable number of patients with CS and CLS who lack mutations in known genes. The study by Bennett et al in this issue of JAMA offers an intriguing explanation for some of these families with PTEN wild-type CS and CLS. It has previously been described that epigenetic inactivation of genes via hypermethylation of their promoters, of other DNA modifications, such as histone deacetylation, or both, can silence tumor suppressor genes. Indeed, silencing of tumor suppressor genes in sporadic cancers by epigenetic regulation is a welldocumented event, and in the case of specific tumor suppressor genes appears to be the more common mechanism of inactivation rather than gene mutation. In some cases, this can occur in the germline, and therefore these epigenetic changes, or “epimutations,” can then be passed to subsequent generations and predispose those individuals to an increased risk of certain cancers. This was the basis of the starting point for Bennett et al wherein they examined the peripheral lymphocytes derived from patients with CS and CLS for hypermethylation of the PTEN promoter. Perhaps not unexpectedly, the investigators did discover, in a significant fraction of these patients, PTEN promoter hypermethylation. However, rather unexpectedly, gene silencing of PTEN was not found, suggesting that methylation of the PTEN promoter did not alter or regulate expression of this tumor suppressor gene. Further study by Bennett el al led to the analysis of a relatively new gene called KILLIN. KILLIN has only recently been identified, and relatively little is known regarding its function or role in human cancers. KILLIN was originally identified as a TP53 regulated inhibitor of DNA synthesis, and

Abstract P4-01-02: Low ATM Expression Levels Predict Resistance to Anthracyclins in TP53 and CHK2 Wild-Type Breast Tumors

Abstract Background: Mutations in TP53 and its upstream p53-activator Chk2, are known to be associated with resistance to anthracycline and mitomycin treatment in locally advanced breast cancer. However, this association is not fully predictive, as some tumours are therapy resistant despite harbouring wild-type TP53 and CHK2. ATM is an upstream activator of both p53 and Chk2. Here, we explored ATM mutational status and expression levels with respect to anthracycline/mitomycine resistance in locally advanced breast cancers having primary chemotherapy with doxorubicin or 5FU-mitomycin and previously analyzed for TP53 and CHK2 status. Methods: In order to investigate ATM's potential predictive role, we performed MLPA-analysis, qPCR on ATM mRNA, ATM promoter methylation analyses and complete sequencing of the entire ATM coding region in patient biopsies from two prospective studies on resistance to doxorubicin and 5-FU/mitomycin in locally advance breast cancer (n=36 and n=38, respectively). Results: We performed complete sequencing of the ATM coding exons in 74 patients including 17 patients revealing primary resistance to chemotherapy. ATM mutations were detected in tumours from 5 patients. No mutation was detected among tumors resistant to chemotherapy. In addition, we observed 9 polymorphic variants present in more than one tumour, affecting or not affecting the amino acid sequence of the ATM protein (n = 34 patients in total). No correlation was observed between polymorphism status and response to therapy. No methylation of the ATM promoter, large deletions or duplication/amplifications of the ATM gene was observed. ATM expression levels varied substantially, with a ratio of 49 between the highest versus lowest level recorded. Among patients with no mutations in TP53 or CHK2, low ATM expression levels were significantly correlated to lack of response to doxorubicin or 5-FU/mitomycin (p=0.023; Mann-Whitney test). In contrast, ATM-levels were not suppressed in non-responding tumors harbouring TP53 or CHK2 mutations (p=0.642; Mann-Whitney test). Defining ATM mRNA levels in the lower 50%, 33% or 25% of the patient cohort to be reduced, tumors with either reduced levels of ATM or mutations affecting TP53 or CHK2 revealed a high risk for therapy resistance (p=0.004, p=0.001 and p=0.006, using the different ATM-level cut-offs; Fischer exact test). Interestingly, in a similar prospective study with a smaller number of patients displaying progressive disease upon treatment with epirubicin (n = 10), we find 2 out 5 TP53 and CHK2-wild-type non-responding tumors to express very low levels of ATM (within the lowest 5% of the cohort). Conclusions: Our data suggests that low expression of ATM may substitute for TP53 and CHK2 mutations causing resistance to anthracycline-and mitomycin therapy in breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-01-02.

S1083 Uncomplicated Peptic Ulcer in the UK: Trends from 1997 to 2005

Background: The duodenal adenomas as seen in familial adenomatous polyposis (FAP) are likely to develop through the classical adenoma-carcinoma pathway as they exhibit mutations in the APC gene, KRAS mutations, p53 dysfunction, and loss of E-cadherin expression. However, it is unknown whether sporadic duodenal adenomas also develop into duodenal carcinoma through the classical adenoma carcinoma sequence. Limited data suggest that sporadic adenomas show similar molecular features as colorectal adenomas, harboring APC and KRAS mutations. Therefore, the aim of the present study was to evaluate the molecular changes of sporadic duodenal adenomas. Methods: Tissue samples of 64 sporadic duodenal adenomas were available from the pathology archives of two academic hospitals. Tissue samples of patients with duodenal carcinoma or patients with FAP were excluded. None of the patients belonged to a known Lynch syndrome family. DNA was extracted from paraffin sections of the adenoma and analyzed for the presence of microsatellite instability. Also, mutation analysis using polymerase chain reaction followed by direct-sequencing was performed. The mutation analysis included mutational hotspots of the Wnt signaling pathway (APC, β-catenin), the MAP kinase pathway (KRAS and BRAF) and the del1100c mutation within the CHK2 gene. Results: Two (3%) of the 64 adenomas showed a high level of MSI (MSI-H). APC mutation analysis was possible in 52 adenomas and revealed a mutation in thirteen (24%), including seven duodenal adenomas with a recurrent 4684insA (K1555fsX1557)mutation. KRASmutation analysis was possible in 52 adenomas and revealed one (2%) mutation (34GGT>TGT (G12C)). β-catenin mutation analysis was possible in 48 adenomas and revealed one (2%) mutation (121ACC>GCC(T41A)). BRAF (58 adenomas) and CHK2 (53 adenomas) mutation analysis did not reveal any mutation. Conclusions: In our cohort of sporadic duodenal adenomas the frequency of Wnt signaling pathway abnormalities is low, most adenomas are microsatellite stable and the number of MAP kinase pathway abnormalities is negligible. In addition, CHK2 mutations seem not to be present in sporadic duodenal adenomas. These findings suggest that a proportion of sporadic duodenal adenomas develop via the same mechanisms (the adenoma-carcinoma sequence) as colorectal adenomas, but it is possible that other molecular changes may also underlie the development of sporadic duodenal adenomas.

S1081 Evaluation of Molecular Changes in Sporadic Duodenal Adenomas

Background: The duodenal adenomas as seen in familial adenomatous polyposis (FAP) are likely to develop through the classical adenoma-carcinoma pathway as they exhibit mutations in the APC gene, KRAS mutations, p53 dysfunction, and loss of E-cadherin expression. However, it is unknown whether sporadic duodenal adenomas also develop into duodenal carcinoma through the classical adenoma carcinoma sequence. Limited data suggest that sporadic adenomas show similar molecular features as colorectal adenomas, harboring APC and KRAS mutations. Therefore, the aim of the present study was to evaluate the molecular changes of sporadic duodenal adenomas. Methods: Tissue samples of 64 sporadic duodenal adenomas were available from the pathology archives of two academic hospitals. Tissue samples of patients with duodenal carcinoma or patients with FAP were excluded. None of the patients belonged to a known Lynch syndrome family. DNA was extracted from paraffin sections of the adenoma and analyzed for the presence of microsatellite instability. Also, mutation analysis using polymerase chain reaction followed by direct-sequencing was performed. The mutation analysis included mutational hotspots of the Wnt signaling pathway (APC, β-catenin), the MAP kinase pathway (KRAS and BRAF) and the del1100c mutation within the CHK2 gene. Results: Two (3%) of the 64 adenomas showed a high level of MSI (MSI-H). APC mutation analysis was possible in 52 adenomas and revealed a mutation in thirteen (24%), including seven duodenal adenomas with a recurrent 4684insA (K1555fsX1557)mutation. KRASmutation analysis was possible in 52 adenomas and revealed one (2%) mutation (34GGT>TGT (G12C)). β-catenin mutation analysis was possible in 48 adenomas and revealed one (2%) mutation (121ACC>GCC(T41A)). BRAF (58 adenomas) and CHK2 (53 adenomas) mutation analysis did not reveal any mutation. Conclusions: In our cohort of sporadic duodenal adenomas the frequency of Wnt signaling pathway abnormalities is low, most adenomas are microsatellite stable and the number of MAP kinase pathway abnormalities is negligible. In addition, CHK2 mutations seem not to be present in sporadic duodenal adenomas. These findings suggest that a proportion of sporadic duodenal adenomas develop via the same mechanisms (the adenoma-carcinoma sequence) as colorectal adenomas, but it is possible that other molecular changes may also underlie the development of sporadic duodenal adenomas.

S1082 Fundic Gland Polyps and Association with Proton Pump Inhibitor Intake: A Prospective Study in 1,780 Upper Gastroduodenal Endoscopies

Background: The duodenal adenomas as seen in familial adenomatous polyposis (FAP) are likely to develop through the classical adenoma-carcinoma pathway as they exhibit mutations in the APC gene, KRAS mutations, p53 dysfunction, and loss of E-cadherin expression. However, it is unknown whether sporadic duodenal adenomas also develop into duodenal carcinoma through the classical adenoma carcinoma sequence. Limited data suggest that sporadic adenomas show similar molecular features as colorectal adenomas, harboring APC and KRAS mutations. Therefore, the aim of the present study was to evaluate the molecular changes of sporadic duodenal adenomas. Methods: Tissue samples of 64 sporadic duodenal adenomas were available from the pathology archives of two academic hospitals. Tissue samples of patients with duodenal carcinoma or patients with FAP were excluded. None of the patients belonged to a known Lynch syndrome family. DNA was extracted from paraffin sections of the adenoma and analyzed for the presence of microsatellite instability. Also, mutation analysis using polymerase chain reaction followed by direct-sequencing was performed. The mutation analysis included mutational hotspots of the Wnt signaling pathway (APC, β-catenin), the MAP kinase pathway (KRAS and BRAF) and the del1100c mutation within the CHK2 gene. Results: Two (3%) of the 64 adenomas showed a high level of MSI (MSI-H). APC mutation analysis was possible in 52 adenomas and revealed a mutation in thirteen (24%), including seven duodenal adenomas with a recurrent 4684insA (K1555fsX1557)mutation. KRASmutation analysis was possible in 52 adenomas and revealed one (2%) mutation (34GGT>TGT (G12C)). β-catenin mutation analysis was possible in 48 adenomas and revealed one (2%) mutation (121ACC>GCC(T41A)). BRAF (58 adenomas) and CHK2 (53 adenomas) mutation analysis did not reveal any mutation. Conclusions: In our cohort of sporadic duodenal adenomas the frequency of Wnt signaling pathway abnormalities is low, most adenomas are microsatellite stable and the number of MAP kinase pathway abnormalities is negligible. In addition, CHK2 mutations seem not to be present in sporadic duodenal adenomas. These findings suggest that a proportion of sporadic duodenal adenomas develop via the same mechanisms (the adenoma-carcinoma sequence) as colorectal adenomas, but it is possible that other molecular changes may also underlie the development of sporadic duodenal adenomas.

Regulation of Chk2 Ubiquitination and Signaling through Autophosphorylation of Serine 379

The Chk2 protein kinase protects genome integrity by promoting cell cycle arrest or apoptosis in response to DNA double-strand breaks, and Chk2 mutations are found in both familial and sporadic cancers. Exposure of cells to ionizing radiation (IR) or radiomimetic drugs induces Chk2 phosphorylation by ATM, followed by Chk2 oligomerization, auto-/transphosphorylation, and activation. Here we demonstrate that Chk2 is ubiquitinated upon activation and that this requires Chk2 kinase activity. Serine 379 (S379) was identified as a novel IR-inducible autophosphorylation site required for ubiquitination of Chk2 by a Cullin 1-containing E3 ligase complex. Importantly, S379 was required for Chk2 to induce apoptosis in cells with DNA double-strand breaks. Thus, auto-/transphosphorylation of S379 is required for Chk2 ubiquitination and effector function.

Comment on the article Genetic contribution to all cancers: the first demonstration using the model of breast cancers from Poland stratified by age at diagnosis and tumour pathology by Lubinski et al., Breast Cancer Res Treat 2008 Apr 15

The study has set an important and complicated goal - to verify the hypothesis that genetic polymorphisms are associated with a predisposition to all malignancies. The article is attractive because of the thorough design, involving a large number of cases and carefully matched controls. The approach by dividing the whole group of breast cancers into 20 subgroups by histological type, grade, and receptor is original and interesting. As it is applied with great care, the data indeed provide novel information about the genetic basis of breast cancer genesis and possibly provide evidence that different types of breast cancer might have different pathogenesis. These data could be helpful in further basic research on breast tumorigenesis, highlighting genetic characteristics for each type of breast cancer. The authors have also shown that heterogeneity of breast cancer at the genetic level is as diverse as the morphological picture, a fact that has to be taken into account in future research design. Comparison of frequencies of polymorphisms in several genes between two groups (cancer patients and controls) in an otherwise homogeneous population leads the authors to several important, and fatalistic, statements: • genetic components play a role in all cancers, • environmental factors act effectively only on predisposed persons. Though the first conclusion seems to be acceptable to a large extent intuitively, the data provided by the authors do not strengthen this belief, at least for us. Thus, 90.6% of cancer patients harboured at least one of the studied markers. But the same is true for more than 80% of the control group. Why in one group are these low and medium penetrance genes considered as factors leading to development of breast cancer (or any cancer in general) and ignored in the other group? What makes a difference between two carriers of e.g. CHK2 mutation? Are there other, as yet unclear, genetic components which increase the significance of this mutation? Or maybe some environmental factors? The authors have taken great pains to stratify the two groups but have ignored any lifestyle aspects. There is no doubt that mutation carriers in genes involved in DNA repair can be affected by a hostile environment more severely and accumulate a greater number of somatic mutations. It is estimated that the TP53 gene is mutated in about 50% of cancers [1]. Should we call such cancer sporadic, or is it genetically predetermined now? Do the authors believe that persons not possessing the listed (and some other) markers could smoke, be obese and enjoy mutagens without being punished? Taking into account the high rate of genetic polymorphisms in the population, the aim of the study - to verify the hypothesis that genetic polymorphisms are associated with a predisposition to all malignancies - is hardly achieved as the relative risk seems to be small and thus other, possibly environmental factors could easily have a greater impact on the outcome in terms of tumour development. The results might reflect, in fact, the genetic variability within the population. According to the results, 9% of breast cancer cases did not show any and 16.6% (162) of control group women were free of any tested. The main clinical consequence of the study in our opinion could be the possibility to release marker negative women from an unnecessary mammographic screening programme. But to receive the ultimate verification of the hypothesis, it will be necessary to perform a follow-up study on this group of women and to wait some 30-40 years. Besides, there is a great probability that during the next years several more cancer predisposing candidate genes will be discovered and it could easily lead to the situation that at least one will be found in 100% of cases and controls. If so, this study approach could be considered as an important contribution to overall understanding about tumorigenesis, but it is difficult to predict its application in clinical practice in the near future.

Cutoff and aubergine Mutations Result in Retrotransposon Upregulation and Checkpoint Activation in Drosophila

Gametogenesis is a highly regulated process in all organisms. In Drosophila, a meiotic checkpoint which monitors double-stranded DNA breaks and involves Drosophila ATR and Chk2 coordinates the meiotic cell cycle with signaling events that establish the axis of the egg and embryo. Checkpoint activity regulates translation of the transforming growth-factor-alpha-like Gurken signaling molecule which induces dorsal cell fates in the follicle cells [1-3]. We found that mutations in the Drosophila gene cutoff (cuff) affect germline cyst development and result in ventralized eggs as a result of reduced Grk protein expression. Surprisingly, cuff mutations lead to a marked increase in the transcript levels of two retrotransposable elements, Het-A and Tart. We found that small interfering RNAs against the roo element are still produced in cuff mutant ovaries. These results indicate that Cuff is involved in the rasiRNA pathway and most likely acts downstream of siRNA biogenesis. The eggshell and egg-laying defects of cuff mutants are suppressed by a mutation in chk2. We also found that mutations in aubergine (aub), another gene implicated in the rasiRNA pathway, are significantly suppressed by the chk2 mutation. Our results indicate that mutants in rasiRNA pathways lead to elevated transposition incidents in the germline, and that this elevation activates a checkpoint that causes a loss of germ cells and a reduction of Gurken protein in the remaining egg chambers.

EDD Mediates DNA Damage-induced Activation of CHK2

EDD, the human orthologue of Drosophila melanogaster "hyperplastic discs," is overexpressed or mutated in a number of common human cancers. Although EDD has been implicated in DNA damage signaling, a definitive role has yet to be demonstrated. Here we report a novel interaction between EDD and the DNA damage checkpoint kinase CHK2. EDD and CHK2 associate through a phospho-dependent interaction involving the CHK2 Forkhead-associated domain and a region of EDD spanning a number of putative Forkhead-associated domain-binding threonines. Using RNA interference, we demonstrate a critical role for EDD upstream of CHK2 in the DNA damage signaling pathway. EDD is necessary for the efficient activating phosphorylation of CHK2 in response to DNA damage following exposure to ionizing radiation or the radiomimetic, phleomycin. Cells depleted of EDD display impaired CHK2 kinase activity and an inability to respond to DNA damage. These results identify EDD as a novel mediator in DNA damage signal transduction via CHK2 and emphasize the potential importance of EDD in cancer.

Mutation analysis of the ATRgene in breast and ovarian cancer families

IntroductionMutations in BRCA1, BRCA2, ATM, TP53, CHK2 and PTEN account for only 20–30% of the familial aggregation of breast cancer, which suggests the involvement of additional susceptibility genes. The ATR (ataxia-telangiectasia- and Rad3-related) kinase is essential for the maintenance of genomic integrity. It functions both in parallel and cooperatively with ATM, but whereas ATM is primarily activated by DNA double-strand breaks induced by ionizing radiation, ATR has been shown to respond to a much broader range of DNA damage. Upon activation, ATR phosphorylates several important tumor suppressors, including p53, BRCA1 and CHK1. Based on its central function in the DNA damage response, ATR is a plausible candidate gene for susceptibility to cancer.MethodsWe screened the entire coding region of the ATR gene for mutations in affected index cases from 126 Finnish families with breast and/or ovarian cancer, 75 of which were classified as high-risk and 51 as moderate-risk families, by using conformation sensitive gel electrophoresis and direct sequencing.ResultsA large number of novel sequence variants were identified, four of which – Glu254Gly, Ser1142Gly, IVS24-48G>A and IVS26+15C>T – were absent from the tested control individuals (n = 300). However, the segregation of these mutations with the cancer phenotype could not be confirmed, partly because of the lack of suitable DNA samples.ConclusionThe present study does not support a major role for ATR mutations in hereditary susceptibility to breast and ovarian cancer.

Defective apoptosis and B-cell lymphomas in mice with p53 point mutation at Ser 23.

Phosphorylation of the p53 tumor suppressor at Ser20 (murine Ser23) has been proposed to be critical for disrupting p53 interaction with its negative regulator, MDM2, and allowing p53 stabilization. To determine the importance of Ser23 for the function of p53 in vivo, we generated a mouse in which the endogenous p53 locus was targeted to replace Ser23 with alanine. We show that, in mouse embryonic fibroblasts generated from Ser23 mutant mice, Ser23 mutation did not dramatically reduce IR-induced p53 protein stabilization or p53-dependent cell cycle arrest. However, in Ser23 mutant thymocytes and in the developing cerebellum, p53 stabilization following IR was decreased and resistance to apoptosis was observed. Homozygous Ser23 mutant animals had a reduced lifespan, but did not develop thymic lymphomas or sarcomas that are characteristic of p53-/- mice. Instead, Ser23 mutant animals died between 1 and 2 years with tumors that were most commonly of B-cell lineage. These data support an important role for Ser20/23 phosphorylation in p53 stabilization, apoptosis and tumor suppression.

Possible causes of chromosome instability: comparison of chromosomal abnormalities in cancer cell lines with mutations in BRCA1, BRCA2, CHK2 and BUB1

A large proportion of epithelial cancers show the chromosome-instability phenotype, in which they have many chromosome abnormalities. This is thought to be the result of mutations that disrupt chromosome maintenance, but the causative mutations are not known. We identified cell lines known to have mutations that might cause chromosome instability, and examined their karyotypes. Two cell lines, the breast cancer line HCC1937 and the pancreatic cancer line CAPAN-1, that have mutations respectively in BRCA1 and BRCA2, had very abnormal karyotypes, with many structural and numerical chromosome changes and substantial variation between metaphases. However, two colorectal cancer lines with mutations in BUB1, a spindle checkpoint protein involved in chromosome segregation, had rather simple near-tetraploid karyotypes, with minimal loss or gain of chromosomes other than the endoreduplication event, and minimal structural change. Apart from tetraploidy, these karyotypes were typical of colorectal lines considered to be chromosomally stable. Two lines derived from the same tumour, DLD-1 and HCT-15, with bi-allelic mutation of CHK2, had karyotypes that were typical of near-diploid colorectal lines considered chromosomally stable. The karyotypes observed supported the proposed role for BRCA1 and BRCA2 mutations in chromosomal instability, but showed that the tested mutations in BUB1 and CHK2 did not result in karyotypes that would have been predicted if they were sufficient for chromosomal instability.

Alteration of G2 Cell Cycle Regulators Occurs during Carcinogenesis of the Endometrium

Objectives: In this study, we examined the alteration of the G2 pathway in endometrial hyperplasia (EH) and endometrioid-type endometrial cancer (EC), and analyzed the relationship between the G2 pathway status and the p53 pathway status. Methods: A total of 103 cases (proliferative phase of the endometrium: 20, EH: 22, and endometrioid-type EC: 61 (I: 39, II: 5, III: 15, recurrence: 2)) were included in this study. We examined the ATM, chk2, CDC25C, cdc2, and cyclin B1 protein expression by immunohistochemistry. In 55 cases (EH: 15; EC: 40), we analyzed chk2 mutations by RT-PCR-SSCP. Results: There were no chk2 mutations in endometrial disease. Elevated or reduced expression rates of ATM, chk2, CDC25C, cdc2 and cyclin B1 were 4.5% (1/22), 0%, 0%, 0% and 4.5% (1/22) in EH and 3.3% (2/61), 4.9% (3/61), 13.1% (8/61), 9.8% (6/61) and 9.8% (6/61) in EC. Alteration of the G2 pathway was higher in EC (32.8%; 20/61) than in EH (9.1%; 2/22; p = 0.047). The G2 pathway was significantly higher in the altered p53 pathway group (48.4%; 15/31) than in the normal p53 pathway group (16.7%; 5/30) in EC (p = 0.0134). The altered p53 pathway tended to be related with the cdc2/cyclin B1 status (p = 0.0529). Conclusions: Alteration of the G2 pathway is thought to occur during carcinogenesis of the endometrium.

The Chk2 Tumor Suppressor Is Not Required for p53 Responses in Human Cancer Cells

Ionizing radiation damages chromosomal DNA and activates p53-dependent transcription in mammalian cells. The Chk2 protein kinase has been hypothesized to be the primary mediator of this response. We have rigorously tested this hypothesis in human cells by disrupting the CHK2 gene through homologous recombination. We found that the p53 response was unexpectedly robust in such cells. Phosphorylation of p53 at serine 20, accumulation of p53 protein, transcriptional activation of p53 target genes, and cell cycle arrest and apoptotic death phenotypes were completely intact regardless of CHK2 status. Our results indicate that Chk2 kinase is not required for p53 activation in human cells and explain why CHK2 and TP53 mutations can jointly occur in human tumors.

The Drosophila chk2 gene loki is essential for embryonic DNA double-strand-break checkpoints induced in S phase or G2.

Cell cycle checkpoints are signal transduction pathways that control the order and timing of cell cycle transitions, ensuring that critical events are completed before the occurrence of the next cell cycle transition. The Chk2 family of kinases is known to play a central role in mediating the cellular responses to DNA damage or DNA replication blocks in various organisms. Here we show through a phylogenetic study that the Drosophila melanogaster serine/threonine kinase Loki is the homolog of the yeast Mek1p, Rad53p, Dun1p, and Cds1 proteins as well as the human Chk2. Functional analyses allowed us to conclude that, in flies, chk2 is involved in monitoring double-strand breaks (DSBs) caused by irradiation during S and G2 phases. In this process it plays an essential role in inducing a cell cycle arrest in embryonic cells. Our results also show that, in contrast to C. elegans chk2, Drosophila chk2 is not essential for normal meiosis and recombination, and it also appears to be dispensable for the MMS-induced DNA damage checkpoint and the HU-induced DNA replication checkpoint during larval development. In addition, Drosophila chk2 does not act at the same cell cycle phases as its yeast homologs, but seems rather to be involved in a pathway similar to the mammalian one, which involves signaling through the ATM/Chk2 pathway in response to genotoxic insults. As mutations in human chk2 were linked to several cancers, these similarities point to the usefulness of the Drosophila model system.

Expression of Chk2 and P53 in Sporadic Breast Cancer

mutations in 7 cases by DNA sequence analysis of the entire coding region. Results: Seven of 30 (23.3%) breast cancers had reduced immuno-expression of Chk2, one of them (1/7, 14.3%) showed a p53 immuno-expression and all of them revealed no Chk2 mutation. Conclusion: Expression of Chk2 protein more reduced in breast cancer with no abnormal p53 immuno-expression. No Chk2 mutation was found in all of Chk2 reduced expression, we hypothesize that there may be a posttranscriptional/ posttranslational mechanism (s) in breast caner to downregulate Chk2 protein expression. (Journal of Korean Breast Cancer Society 2003;6:141-147)

Mutation analysis of the CHK2 gene in breast carcinoma and other cancers.

Mutations in the CHK2 gene at chromosome 22q12.1 have been reported in families with Li-Fraumeni syndrome. Chk2 is an effector kinase that is activated in response to DNA damage and is involved in cell-cycle pathways and p53 pathways. We screened 139 breast tumors for loss of heterozygosity at chromosome 22q, using seven microsatellite markers, and screened 119 breast tumors with single-strand conformation polymorphism and DNA sequencing for mutations in the CHK2 gene. Seventy-four of 139 sporadic breast tumors (53%) show loss of heterozygosity with at least one marker. These samples and 45 tumors from individuals carrying the BRCA2 999del5 mutation were screened for mutations in the CHK2 gene. In addition to putative polymorphic regions in short mononucleotide repeats in a non-coding exon and intron 2, a germ line variant (T59K) in the first coding exon was detected. On screening 1172 cancer patients for the T59K sequence variant, it was detected in a total of four breast-cancer patients, two colon-cancer patients, one stomach-cancer patient and one ovary-cancer patient, but not in 452 healthy individuals. A tumor-specific 5' splice site mutation at site +3 in intron 8 (TTgt [a --> c]atg) was also detected. We conclude that somatic CHK2 mutations are rare in breast cancer, but our results suggest a tumor suppressor function for CHK2 in a small proportion of breast tumors. Furthermore, our results suggest that the T59K CHK2 sequence variant is a low-penetrance allele with respect to tumor growth.