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

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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.
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Research article
Mutation analysis of the CHK2 gene in breast carcinoma and
other cancers
Sigurdur Ingvarsson
1
, Bjarnveig I Sigbjornsdottir
2
, Chen Huiping
2
, Sigridur H Hafsteinsdottir
2
,
Gisli Ragnarsson
2
, Rosa B Barkardottir
2
, Adalgeir Arason
2
, Valgardur Egilsson
2
and Jon Th Bergthorsson
2
1
Institute for Experimental Pathology, University of Iceland, Reykjavik, Iceland
2
Department of Pathology, University Hospital, Reykjavik, Iceland
Correspondence: Sigurdur Ingvarsson, Institute for Experimental Pathology, University of Iceland, Keldur v/Vesturlandsveg, 112 Reykjavik, Iceland.
Tel: +354 5674700; fax: +354 5673979; e-mail: siguring@hi.is
Introduction
Chk2 (Cds1) is a protein kinase that is involved in cell-
cycle checkpoint control by phosphorylating Cdc25 phos-
phatases, which subsequently results in their inhibition (i.e.
degradation or export from the nucleus) [1–4]. Other sub-
strates of Chk2 are p53 and Brca1, which are involved in
cell-cycle control, apoptosis, and DNA repair [4–7]. The
serine 20 of p53 is phosphorylated by Chk2, and thereby
interrupts the binding of p53 to Mdm2 and interrupts p53
ubiquitination, resulting in greater stability of p53 [8].
Chk2 is activated on DNA damage by phosphorylation
signaling from the Atm kinase [8–13].
LOH = loss of heterozygosity; PCR = polymerase chain reaction; RT = reverse transcriptase; SSCP = single-strand conformation polymorphism.
Available online http://breast-cancer-research.com/content/4/3/R4
Abstract
Background: 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.
Methods: 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.
Results: 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.
Conclusion: 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.
Keywords: breast cancer, CHK2, chromosome 22q, mutation, polymorphism
Received: 21 September 2001
Revisions requested: 6 November 2001
Revisions received: 3 January 2002
Accepted: 26 February 2002
Published: 20 March 2002
Breast Cancer Res 2002, 4:R4
This article may contain supplementary data which can only be found
online at http://breast-cancer-research.com/content/4/3/R4
© 2002 Ingvarsson et al., licensee BioMed Central Ltd
(Print ISSN 1465-5411; Online ISSN 1465-542X)
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Breast Cancer Research Vol 4 No 3 Ingvarsson et al.
Germ line mutations have been detected in the TP53 and
CHK2 genes in patients with Li–Fraumeni syndrome
[14–16]. These mutated forms of the Chk2 may have dis-
abilities in protein–protein interactions and in being phos-
phorylated by Atm [17]. Characteristic tumor types in
patients with Li–Fraumeni syndrome are breast cancer,
sarcoma, brain carcinoma and adrenal cortex carcinoma,
and multiple primary tumors can be observed in the same
individuals. There are few reports on somatic mutations of
CHK2 in tumors, but mutations have been found in a
colon carcinoma cell line and in a case of primary small-
cell lung cancer, lymphoma and myelodysplastic syn-
drome, respectively [14,18–20]. We used microsatellite
markers to analyse the loss of heterozygosity (LOH) at
chromosome region 22q, where the CHK2 gene is
located, and screened breast and other tumors for muta-
tions in the CHK2 gene.
Materials and methods
Primary breast carcinoma tissue was obtained on the day of
surgery. Blood samples from the patients were collected in
EDTA and, if not processed immediately, tumor and blood
were quick-frozen at –70°C. LOH at chromosome 22q was
analysed using seven microsatellite markers on 139 spo-
radic breast tumors. DNA was analysed by PCR primers
that amplify markers D22S277, D22S283, D22S1177,
D22S272, D22S423, D22S1179 and D22S282. These
markers map telomeric to CHK2 (at position 25,750 kb), or
at positions 32,830 kb, 33,300 kb, 33,800 kb, 35,600 kb,
36,900 kb, 40,100 kb and 40,350 kb, respectively.
DNA samples (25 ng) were subjected to PCR analysis in a
total volume of 25 µl using DynaZyme™ polymerase
(Finnzymes Oy, Espoo, Finland), in 120 µM of each
deoxynucleotide triphosphate and 0.24 µM primers. After
5 min of denaturation at 94°C, samples were subjected to
35 cycles of amplification, consisting of 30 s at 94°C, 30 s
at 55°C and 1 min at 72°C. The PCR products were
denatured in formamide buffer, separated on 6.5% poly-
acrylamide denaturing gels, and transferred to a Hybond-
N
+
nylon membrane (Amersham, Aylesbury, UK).
Hybridisation of a peroxidase-labeled probe to the PCR
products was visualised using the enhanced chemilumi-
nescence labeling method (ECL kit; Amersham).
LOH was evaluated visually by comparing the intensity of
alleles from normal and tumor DNA. The absence or
decrease in the intensity of one allele relative to the other
was considered as LOH. Tumor samples were scored for
LOH at chromosome 22q if at least one informative marker
showed LOH. One sample that was homozygous for all
tested markers was excluded from the study, while other
samples tested heterozygous with two to seven markers.
Sporadic tumors (BRCA1 and BRCA2 mutation carriers
were excluded) showing LOH at chromosome 22q (74
cases) and 45 breast tumors from carriers of the BRCA2
999del5 mutation [21], not analysed for LOH at 22q, were
analysed with single-strand conformation polymorphism
(SSCP) and DNA sequencing, using 17 primers for all of
the 15 exons of the CHK2 gene. The set of BRCA2
samples was screened to address the possibility that
germ line variants could modify BRCA2 risk. The primers
were ordered from TAG Copenhagen A/S and information
on them is presented in Table 1. The SSCP and DNA
sequencing conditions were as described earlier [22].
An additional 1098 tumors of various origins were
analysed for the detected T59K sequence variant using
the primers from exon 2 and DNA sequencing. RNA was
isolated from a tumor showing a splice site mutation and
analysed with RT-PCR using primers from exon 7
(forward, 5-CCCAGCTCTCAATGTTGAAACAG-3) and
exon 9 (reverse, 5-CTGCACAGCCAAGAGCATCTGG-3).
Abnormally sized PCR products were cut from 4%
agarose gels and DNA sequenced.
The chi-squared test and Fisher’s exact test were used to
compare differences of the frequency of sequence vari-
ants, between controls, individuals with sporadic breast
cancer and individuals with breast cancer carrying the
BRCA2 999del5 germ line mutation. The research plan
was approved by the National Bioethics Committee.
Results
Seventy-four out of 139 (53%) sporadic breast tumors were
detected with LOH at chromosome 22q. Only 11 (16%) of
the tumors with 22q LOH showed an almost complete loss
of the alleles, while other tumors showed less decrease in
allele intensity, presumably due to contamination of DNA
from non-malignant cells. To evaluate whether CHK2
sequence variants could have modifying effects on the phe-
notype of BRCA2 mutation carriers, these 74 samples and
45 breast tumors from individuals carrying the BRCA2
999del5 mutation were screened for mutations in the
CHK2 gene using SSCP and DNA sequencing.
Five sequence variants were detected: four in the group of
74 sporadic tumors and three in the BRCA2 999del5
group, with two variants common to both groups (Tables 2
and 3). Four of these five sequence variants were also
detected in the blood of the patients, indicating a germ line
variant, and one mutation was tumor specific, a +3TTgt(a
c)agt in the 5 splice site of intron 8, detected in the
group of sporadic breast cancer. This somatic mutation
was detected in the tumor of an individual diagnosed with
breast cancer at the age of 45 years. LOH at 22q in this
tumor was evident from the microsatellite marker analysis.
This position in the splice site at the exon 8–intron 8
boundary does not include the part with the highest con-
servation, but we analysed the RNA from the tumor to see
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whether splicing was altered. RT-PCR analysis of RNA
from this tumor showed one extra band of lower molecular
weight and one band of higher molecular weight than the
wild-type transcript, not detected in five nonmalignant and
five breast cancer tissues (data not shown). DNA
sequencing of the RT-PCR product of the smaller sized
transcript showed that the exon 8 sequence was missing.
One of the sequence variants was a clear polymorphism
of A G at nucleotide 252, not affecting the correspond-
ing codon 84 for glutamic acid. We also detected a
sequence variant in a non-coding exon of the CHK2 gene,
a deletion of a thymine. This occurred in a short stretch of
Available online http://breast-cancer-research.com/content/4/3/R4
Table 1
Information on the primers used for single-strand conformation polymorphism and DNA sequencing of the CHK2 gene
Exon number Forward primer Reverse primer
15-GGGTTTTGATTGGCTGAGG-3 5-GCTCAAAACTACAGACAAAGC-3
25-CTCACCTTTGTTGTTGGACAC-3 5-GGACACTGTCTCTAAGGAGC-3
25-AGTCCTCTCACTCCAGC-3 5-ATCAGAACCTTCCACCTGG-3
35-ATTCAACAGCCCTCTGATGC-3 5-CAGCTCTCCTAGATACATGG-3
45-TCTGCTATTCAAAGTCTG-3 5-TCCTCCTATGAGAGAGTGG-3
55-GAAATGAGAAACCACCAATCAC-3 5-TCAGTGATCGCCTCTTGTG-3
65-TACTTGAAGTGGACCCAGG-3 5-GGGAAGTTATGAAGACGTG-3
75-CAAAGTGCTAGGGTTACAGG-3 5-CAGCCTTGAGTCAACTGAG-3
85-GCTCTTGTGGTTTTCCTCTTGG- 5-CCTACATTAGATTCTTTGGTGG-3
95-CTGTCCAAGTGCGTTTTCC-3 5-CGATTTCTGCCTAATTCAGGG-3
10 5-ACGGCTTACGGTTTCACC-3 5-CAAGAATCTACAGGAATAGCC-3
11 5-CTTGGACTGGCAGACTATG-3 5-CTCCTACCAGTCTGTGCAG-3
12 5-ATGCCACTGAGAATGCCAC-3 5-CTCCCACCACAGCACATAC-3
13 5-CCTTTTCACTGTGATTTGCCC-3 5-CATGTTTCTGTCCTCTGTCTC-3
14 5-CTAGCCCTGTCATTCTAGG-3 5-CTCCTTAAGCCCAGACTAC-3
15 5-TGTGTTGTGAACTCCGTGG-3 5-CAGAGTGAGACTCCATCTC-3
15 5-CTTTACTGGAAGCATATTGAGG–3 5-AGATGACAGAGTGAAAGAAGG-3
Table 2
CHK2 sequence variations in breast cancer
Sequence variation Codon Nucleotide change and position Effect on protein
Germ line 59 C176A T59K
Non-coding delT (exon 1) None
Non-coding insA (intron 2) None
Somatic 303 +3TTgt(a c)agt (intron 8) Splice site
Polymorphism 84 A252G None
All mutations were heterozygous. The total number of patients was 119 (74 sporadic and 45 BRCA2 999del5).
Table 3
Frequency of the detected sequence variants of the CHK2 gene
in breast cancer and controls
Sequence variant Controls Sporadic BRCA2 999del5
T59K 0/904 1/146 0/74
Exon 1 (delT) 5/328 1/148 2/90
Intron 2 (insA) 38/344 21/130 14/78
A252G 5/344 0/130 1/74
The numbers stated refer to the number of chromosomes analysed.
mononucleotide repeats of four thymines. An additional
sequence variant was detected in intron 2, an insertion of
an adenine in a mononucleotide repeat of five adenines. A
missense mutation at codon 59 was also detected, which
substituted lysine for threonine.
The frequency of these sequence variants was estimated
in individuals without previous diagnosis of cancer, in indi-
viduals with sporadic breast cancer, and in individuals with
breast cancer carrying the BRCA2 999del5 mutation
(Table 3). Chi-squared analysis and Fisher’s exact tests
did not result in significant difference between the three
groups for any of the sequence variants.
The only variant allele not appearing in normal control was
the T59K mutation, and this was analysed further in addi-
tional controls (in total, 452 individuals) and in individuals
diagnosed with cancer in the breast and other tissues (in
total, 1172 individuals diagnosed with cancer) (Table 4).
The T59K sequence variant was found in additional
patients with cancer of the breast (four individuals, one
thereof bilateral), cancer of the colon and ovary (one indi-
vidual), cancer of the colon (one individual), and cancer of
the stomach (one individual). All individuals who were
detected with the CHK2 T59K mutation in this screening
are females.
Four of the CHK2 T59K carriers were found to be
members of two cancer families. One of the two individu-
als with colorectal cancer is a sister of the individual with
gastric cancer. A third member of this family (a nephew of
the two aforementioned cases) also developed colon
cancer, but was not a carrier of the CHK2 T59K sequence
variant. However, a distant relative with prostate cancer
was a carrier of the T59K sequence variant.
Two of the breast-cancer cases (diagnosed at 29 years
and 45 years, respectively) were first cousins in a previ-
ously reported cancer family [23]. The third cancer case in
this family (stomach cancer diagnosed at the age of
55 years and thyroid cancer diagnosed at the age of
58 years in the same individual), a brother of the breast-
cancer case diagnosed at age 45 years, is a T59K carrier,
but two individuals within this family (with breast cancer
and thyroid cancer, respectively) were not T59K carriers.
In addition, six healthy individuals of this family did not
carry the T59K sequence variant. The two breast-cancer
cases with the T59K sequence variant, but no obvious
family history of cancer, were diagnosed at 83 years (a
bilateral case) and 42 years of age, respectively.
In summary, the CHK2 T59K sequence variant was
detected in seven individuals by the screening (including
the original carrier from Table 2) and in an additional two
individuals in the family analysis, producing a total of nine
individuals. Three out of these nine individuals were diag-
nosed with two tumors (i.e. in total, 12 tumors in nine indi-
viduals). Eleven out of the 12 tumors were available for
LOH analysis at chromosome 22q, using markers
D22S277, D22S1177, D22S272, D22S423 and
D221179. Eight of these 11 tumors showed LOH with at
least one marker. The three tumors not showing LOH
derived from breast cancer (an individual with bilateral
disease had LOH at chromosome 22q in only one of the
two primary tumors).
Discussion
This screening of a large number of tumor samples sug-
gests that CHK2 gene inactivation does not play a major
role in the pathogenesis of cancer growth. We identified a
somatic mutation at a splice site in one tumor sample,
resulting in abnormal splicing of the gene, in an individual
diagnosed with breast cancer at the age of 45 years. The
wild-type copy of the CHK2 gene was lost in this individ-
ual, suggesting a typical two-hit mechanism of a tumor
suppressor gene.
Only one of the four detected germ line variants affects
the Chk2 protein sequence, by substituting lysine for
threonine at amino acid position 59. This region of the
protein is poorly characterised and the functional aspects
of the threonine in this position are unclear. This position
is close to an Atm phosphorylation site at T68, but there is
no evidence so far for a phosphorylation at T59 [9,13].
The T59K sequence variant is not likely to be highly pene-
trant with respect to tumor growth. Seven of the nine indi-
Breast Cancer Research Vol 4 No 3 Ingvarsson et al.
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Table 4
Frequency of the CHK2 T59K mutation in nine Icelandic
groups
a
Patient group Number tested Carriers %
Breast cancer 685
b
4
c
0.6
Colorectal cancer 119 2
d
1.6
Lung cancer 74 0 0
Renal cell carcinomas 71 0 0
Stomach cancer 37 1 2.7
Ovarian cancer 43 1
d
2.3
Testis cancer 31 0 0
Other cancers 112 0 0
Controls 452 0 0
a
The different groups consist of unselected patients, except in the
case of breast cancer where patients with BRCA2 999del5 have been
omitted.
b
Including samples from Table 3.
c
One of these individuals is
a bilateral case.
d
The total number of carriers in this screening is seven,
but one individual is a carrier of both colon and ovarian cancer and
therefore appears twice in the table.
viduals with the T59K sequence variant were diagnosed
with more than one primary tumor and/or are members of
cancer families. The remaining two individuals carrying the
CHK2 T59K mutation were diagnosed with breast cancer
at the age of 42 years and colon cancer at the age of
66 years, respectively. The absence of complete segrega-
tion in one family with a history of colon cancer and a
second family with breast cancer (in addition to other
tumor types), and the low frequency of mutation in individ-
uals with various tumor types, do not support the idea of a
highly penetrant germ line variant, but the modifying effect
on tumor pathogenesis may be of relevance.
We suggest that the T59K germ line variant is a low-pene-
trance allele with respect to tumor growth, but additional
genetic variations of unknown origin may enhance the
family history of cancer. Even though an amino acid is
changed as a result of the sequence variation, this study
does not clearly show, but does support, a dysfunctional
variant. As suggested by the absence of sequence vari-
ants in the CHK2 gene in individuals carrying the BRCA2
999del5 mutation, there is no evidence of CHK2 acting as
a modifying genetic factor on the tumor phenotype in
these individuals. The remaining three germ line variants of
CHK2 are also detected in the normal population and are
therefore putative polymorphisms. The silent polymor-
phism at codon 84 has been reported earlier in other pop-
ulations [14,18], while the other two polymorphisms at a
short mononucleotide stretch are novel.
The CHK2 gene is known to have several genomic copies
through the genome that have a high sequence conserva-
tion [24]. Our sequence comparison indicates that the
first 10 exons do not give problems due to DNA sequence
homology between genes, but homology is detected for
exon 11 to exon 14 of the CHK2 gene. In our primer set,
only primers from exon 11 give a perfect match with an
additional CHK2 copy from chromosome 10. Other
primers for exons 12 to 14 have one to three nucleotide
mismatches. It is therefore unlikely that the detected vari-
ants are from other copies of the CHK2 gene, since they
are not from the highly conserved part of the gene. There
is a risk that we are missing sequence variants from exons
11 to 14, although they could be detected as bands with
weak intensity. It is therefore difficult to rely on this work
being a full screen of CHK2 sequence variants.
In conclusion, CHK2 germ line mutations and somatic
mutations are detected in breast cancer, but CHK2 inacti-
vation does not play a major role in the cancer growth.
Somatic mutations in the CHK2 gene are rare in breast
tumors. The detected CHK2 T59K germ line allele is prob-
ably of low penetrance with respect to cancer growth. In
breast cancer, mutations in TP53 and CHK2 genes proba-
bly explain only a part of the genetic instability detected in
breast tumors.
Acknowledgements
This work was funded by the Icelandic Cancer Society and the Univer-
sity of Iceland Research Fund.
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... The CHK2 gene encodes the CHK2 kinase that plays a key role in the cellular response to DNA damage and the regulation of the cell cycle through phosphorylation of CDC25 phosphatases [102]. It is activated in response to DSB emergence, ionizing radiation, or other genotoxic factors. ...
... According to some researchers, the rs17879961 polymorphism may be a founding mutation in various ethnic populations. Polymorphisms in the CHK2 gene have also been analyzed in patients with other cancers, including breast cancer, and a variant was identified that was more common in cancer patients than in healthy people, suggesting that CHK2 polymorphisms may be associated with cancer risk [102]. We found no studies examining CHK2 gene polymorphisms in other populations. ...
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The incidence of thyroid cancer, one of the most common forms of endocrine cancer, is increasing rapidly worldwide in developed and developing countries. Various risk factors can increase susceptibility to thyroid cancer, but particular emphasis is put on the role of DNA repair genes, which have a significant impact on genome stability. Polymorphisms of these genes can increase the risk of developing thyroid cancer by affecting their function. In this article, we present a concise review on the most common polymorphisms of selected DNA repair genes that may influence the risk of thyroid cancer. We point out significant differences in the frequency of these polymorphisms between various populations and their potential relationship with susceptibility to the disease. A more complete understanding of these differences may lead to the development of effective prevention strategies and targeted therapies for thyroid cancer. Simultaneously, there is a need for further research on the role of polymorphisms of previously uninvestigated DNA repair genes in the context of thyroid cancer, which may contribute to filling the knowledge gaps on this subject.
... Haruki et al reported a low frequency of somatic mutations in the CHEK2 gene in small-cell lung cancer (16). As for breast cancer, some studies reported a high frequency of CHEK2 mutations, while others found the contrary (17). CHEK2 mutations were also reported in a subset of osteosarcomas (18). ...
... Bell et al suggested a possible role of CHEK2 in cancer development rather than a tumor suppressor gene (39). The 1157T, 1100delC and 1422delT mutations in CHEK2 are the most commonly reported mutations in different cancers, including Li-Fraumeni syndrome, breast cancer and prostate cancer (17,39,40). Collectively, most studies supported the association between the occurrence of CHEK2 mutations/expression and the development of certain cancers. ...
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Background: Prostate cancer (PCa) is one of the most common types of cancer among men. Mutations and accumulation of chromosomal deviations are correlated with the development and aggressiveness of PCa. Cell cycle checkpoint pathways and DNA repair mechanisms are reported to deviate in cancers. Mammalian checkpoint kinase 1/2 (CHEK1/CHEK2) genes act as key signal transducers inside the genomic integrity checkpoints. CHEK1 and CHEK2 gene mutations were reported in a few different types of cancers. In PCa, CHEK2 mutations were studied, but CHEK1 gene variations were not well investigated. Objective: This study aimed to investigate the occurrence of variations in the CHEK1 and CHEK2 genes in PCa in the Jordanian population. Methods: Formalin-fixed paraffin-embedded PCa specimens of radical prostatectomy surgical procedures from 74 Jordanian patients were subjected to DNA extraction, polymerase chain reactions and Sanger sequencing to screen the mutations in selected exons of CHEK1 and CHEK2 tumor suppressor genes. Results: The presence of F281L (T/C) (1.4%) homologous missense point mutation in the kinase domain of the CHEK2 gene and P188P (1.4%) silent point mutation in the kinase domain of the CHEK1 gene. In addition, the 1100delC mutation was not detected in the studied PCa specimens. Conclusion: In line with previous reports, the presence of CHEK2 mutation with a frequency of 1.4% supported the possible role of genetic variants of this gene in the development of PCa. No 1100delC mutation was detected in this study. No association was found in this study between CHEK1 mutations and the development of PCa. Further studies are needed with larger cohorts along with a screening of more exons in order to shed more light on the frequency of CHEK2 gene mutations and their role in the development of PCa in Jordan.
... The growing body of evidence that associates CHEK2 with breast cancer has led to increased genetic testing of CHEK2, and as a consequence to the identification of more (rare) genetic variants in this gene for which clinical significance is unknown (11)(12)(13)(14)(15). In fact, 1,332 variants of uncertain significance (VUS) in CHEK2 have currently been reported in ClinVar (as of October 2021; ref. 16), most of which (i.e., 1,139) are missense variants. ...
Article
Circular RNAs (circRNA) and N6-methyladenosine (m6A) modification are extensively involved in the progression of diverse tumors, including hepatocellular carcinoma (HCC). However, the cross-talk between circRNAs and m6A remains elusive in the pathogenesis of HCC. Here we investigated m6A-mediated regulation of circRNAs in HCC. m6A-related circRNAs were identified by integrating information from two published studies, revealing circular cleavage and polyadenylation specific factor 6 (circCPSF6) as a novel m6A-modified circRNA. circCPSF6 was dominated by ALKBH5-mediated demethylation, followed by the recognization and destabilization by YTHDF2. Meanwhile, circCPSF6 was upregulated in HCC specimens, and elevated circCPSF6 expression served as an independent prognostic factor for worse survival of patients with HCC. Loss-of-function assays demonstrated that circCPSF6 maintained cell proliferation and tumorigenicity and reinforced cell motility and tumor metastasis. circCPSF6 triggered expression of YAP1, further activating its downstream cascade. Mechanistically, circCPSF6 competitively bound PCBP2, blunting its binding to YAP1 mRNA, thereby sustaining the stability of YAP1. Functionally, removal of YAP1 reversed the effects of circCPSF6 in vitro and in vivo. Aberrant activation of the circCPSF6-YAP1 axis promoted HCC malignancy. These findings offer novel insights into the regulation of circRNAs by m6A modifications and the role of this epigenetic reprogramming in HCC. Significance: This study advances the understanding of the interplay between m6A methylation and circRNAs in hepatocellular carcinoma, highlighting the potential of circCPSF6 as a therapeutic target.
... The growing body of evidence that associates CHEK2 with breast cancer has led to increased genetic testing of CHEK2, and as a consequence to the identification of more (rare) genetic variants in this gene for which clinical significance is unknown (11)(12)(13)(14)(15). In fact, 1332 variants of uncertain significance (VUS) in CHEK2 have currently been reported in ClinVar (16) (as of October 2021), most of which (i.e., 1139) are missense variants. ...
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Heterozygous carriers of germline loss-of-function variants in the tumor suppressor gene checkpoint kinase 2 (CHEK2) are at an increased risk for developing breast and other cancers. While truncating variants in CHEK2 are known to be pathogenic, the interpretation of missense variants of uncertain significance (VUS) is challenging. Consequently, many VUS remain unclassified both functionally and clinically. Here we describe a mouse embryonic stem (mES) cell-based system to quantitatively determine the functional impact of 50 missense VUS in human CHEK2. By assessing the activity of human CHK2 to phosphorylate one of its main targets, Kap1, in Chek2 knockout mES cells, 31 missense VUS in CHEK2 were found to impair protein function to a similar extent as truncating variants, while 9 CHEK2 missense VUS resulted in intermediate functional defects. Mechanistically, most VUS impaired CHK2 kinase function by causing protein instability or by impairing activation through (auto)phosphorylation. Quantitative results showed that the degree of CHK2 kinase dysfunction correlates with an increased risk for breast cancer. Both damaging CHEK2 variants as a group [OR 2.23; 95% confidence interval (CI), 1.62-3.07; P < 0.0001] and intermediate variants (OR 1.63; 95% CI, 1.21-2.20; P = 0.0014) were associated with an increased breast cancer risk, while functional variants did not show this association (OR 1.13; 95% CI, 0.87-1.46; P = 0.378). Finally, a damaging VUS in CHEK2, c.486A>G/p.D162G, was also identified, which cosegregated with familial prostate cancer. Altogether, these functional assays efficiently and reliably identified VUS in CHEK2 that associate with cancer. Significance: Quantitative assessment of the functional consequences of CHEK2 variants of uncertain significance identifies damaging variants associated with increased cancer risk, which may aid in the clinical management of patients and carriers.
... We also found that FadA enhanced E-cadherin/βcatenin activation to upregulate chk2 in turn, thereby inducing DNA damage in CRC cells. Chk2 has been implicated in other cancers as well, such as breast cancer [46]. In CRC, the involvement of chk2 is also well-documented [47][48][49][50]. ...
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Background: Globally, colorectal cancer (CRC) affects more than 1 million people each year. In addition to non-modifiable and other environmental risk factors, Fusobacterium nucleatum infection has been linked to CRC recently. In this study, we explored mechanisms underlying the role of Fusobacterium nucleatum infection in the progression of CRC in a mouse model. Methods: C57BL/6 J-Adenomatous polyposis coli (APC) Min/J mice [APC (Min/+)] were treated with Fusobacterium nucleatum (109 cfu/mL, 0.2 mL/time/day, i.g., 12 weeks), saline, or FadA knockout (FadA-/-) Fusobacterium nucleatum. The number, size, and weight of CRC tumors were determined in isolated tumor masses. The human CRC cell lines HCT29 and HT116 were treated with lentiviral vectors overexpressing chk2 or silencing β-catenin. DNA damage was determined by Comet assay and γH2AX immunofluorescence assay and flow cytometry. The mRNA expression of chk2 was determined by RT-qPCR. Protein expression of FadA, E-cadherin, β-catenin, and chk2 were determined by Western blot analysis. Results: Fusobacterium nucleatum treatment promoted DNA damage in CRC in APC (Min/+) mice. Fusobacterium nucleatum also increased the number of CRC cells that were in the S phase of the cell cycle. FadA-/- reduced tumor number, size, and burden in vivo. FadA-/- also reduced DNA damage, cell proliferation, expression of E-cadherin and chk2, and cells in the S phase. Chk2 overexpression elevated DNA damage and tumor growth in APC (Min/+) mice. Conclusions: In conclusion, this study provided evidence that Fusobacterium nucleatum induced DNA damage and cell growth in CRC through FadA-dependent activation of the E-cadherin/β-catenin pathway, leading to up-regulation of chk2.
... e most frequent mutations associated with hereditary cancer include those that affect DNA damage repair (DDR) genes, the most important of which are mutations in the BRCA1, BRCA2, and TP53 genes [5]. Sporadic cancer represents approximately 85% of all cases of breast cancer and is associated with some of the risk factors mentioned above; however, it has also been associated with exposure to carcinogens, such as air pollutants [6], electromagnetic radiation [7], and DDR gene expression dysregulation [8]. ...
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Breast cancer is the cancer with the highest prevalence in women and is the number-one cause of cancer mortality worldwide. Cell transduction is a fundamental process in the development and progression of cancer. Modifications in various cell signalling pathways promote tumour cell proliferation, progression, and survival. The PI3K/Akt/mTOR pathway is an example of that, and it is involved in growth, proliferation, survival, motility, metabolism, and immune response regulation. Activation of this pathway is one of the main causes of cancer cell resistance to antitumour therapies. This makes PI3K/Akt/mTOR signalling a crucial object of study for understanding the development and progression of this disease. Thus, this pathway may have a role as a potential therapeutic target, as well as prognostic and diagnostic value, in patients with breast cancer. Despite the existence of selective PI3K/Akt/mTOR pathway inhibitors and current clinical trials, the cellular mechanisms are not yet known. The present review aims to understand the current state of this important disease and the paths that must be forged.
... 41 The loss of heterozygosity at the CHK2 gene (at chromosome 22q) was associated with 53% of the breast cancers. 42 In Saudi Arabia, the prevalence of TP53 gene mutation was found to be 40%, which lies amongst the highest values worldwide, and about 73% of these patients with TP53 genes mutations were found with < 50 years of age. 43 However, this high prevalence is due to somatic alterations, notgermline mutations. ...
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In recent years there have been important advances in molecular genetics and linkage analysis of the breast cancer. Beside germline BRCA1 or BRAC2 mutations, and somatic genetic alterations, epigenetic alterations in numerous genes play an essential role in the tumorigenesis of breast cancer. TP53, STK11, PTEN, CDH1, NF1 or NBN mutations are associated with high breast cancer associated syndromes. Mutations in DNA repair associated genes (ATM, CHEK2, BRIP1, PALB2 and RAD50) are associated with increased breast cancer risk. Moreover, several single nucleotide polymorphisms (SNPs) were considered as breast cancer susceptibility polymorphisms within genes (FGFR2, TOX3, LSP1, MAP3K1, and TGFB1). This review discusses breast cancer genetic susceptibility, highlights recent advances in breast cancer genetics, with a particular focus in Saudi women.
... In the present study, it was revealed that depletion of PRNCR1-2 markedly altered the phosphorylation of AKT and CHK2, whereas total AKT and CHK2 protein levels were not affected. CHK2 acts as an important regulator of cell cycle progression and proliferation (27,28), and CHK2 signaling is activated by phosphorylation of itself and downstream substrates (29). AKT is another key regulator of tumor cell proliferation, cell cycle progression, migration and invasion (30)(31)(32), which is also activated by its phosphorylation (33). ...
... CHEK2 activation in response to DNA damage induces a cell cycle checkpoint [71]. CHEK2 variants predispose individuals to breast and colon cancer [72] and it has been shown to be a negative regulator of prostate cancer growth [73]. RAD50 is a member of the MRN (MRE11-RAD50-NBS1) complex which functions as a scaffold for sensing DNA damage [74]. ...
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Background The cellular effects of androgen are transduced through the androgen receptor, which controls the expression of genes that regulate biosynthetic processes, cell growth, and metabolism. Androgen signaling also impacts DNA damage signaling through mechanisms involving gene expression and transcription-associated DNA damaging events. Defining the contributions of androgen signaling to DNA repair is important for understanding androgen receptor function, and it also has translational implications. Methods We generated RNA-seq data from multiple prostate cancer lines and used bioinformatic analyses to characterize androgen-regulated gene expression. We compared the results from cell lines with gene expression data from prostate cancer xenografts, and patient samples, to query how androgen signaling and prostate cancer progression influences the expression of DNA repair genes. We performed whole genome sequencing to help characterize the status of the DNA repair machinery in widely used prostate cancer lines. Finally, we tested a DNA repair enzyme inhibitor for effects on androgen-dependent transcription. Results Our data indicates that androgen signaling regulates a subset of DNA repair genes that are largely specific to the respective model system and disease state. We identified deleterious mutations in the DNA repair genes RAD50 and CHEK2. We found that inhibition of the DNA repair enzyme MRE11 with the small molecule mirin inhibits androgen-dependent transcription and growth of prostate cancer cells. Conclusions Our data supports the view that crosstalk between androgen signaling and DNA repair occurs at multiple levels, and that DNA repair enzymes in addition to PARPs, could be actionable targets in prostate cancer. Electronic supplementary material The online version of this article (10.1186/s12885-018-4848-x) contains supplementary material, which is available to authorized users.
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Cancer is a disease caused by the accumulation of genetic and epigenetic changes in two types of genes: tumor suppressor genes (TSGs) and proto-oncogenes. Extensive research has been conducted over the last few decades to elucidate the role of TSGs in cancer development. In cancer, loss of TSG function occurs via the deletion or inactivation of two alleles, according to Knudson’s two-hit model hypothesis. It has become clear that mutations in TSGs are recessive at the level of an individual cell; therefore, a single mutation in a TSG is not sufficient to cause carcinogenesis. However, many studies have identified candidate TSGs that do not conform with this standard definition, including genes inactivated by epigenetic silencing rather than by deletion. In addition, proteasomal degradation by ubiquitination, abnormal cellular localization, and transcriptional regulation are also involved in the inactivation of TSGs. This review incorporates these novel additional mechanisms of TSG inactivation into the existing two-hit model and proposes a revised multiple-hit model that will enable the identification of novel TSGs that can be used as prognostic and predictive biomarkers of cancer.
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In response to DNA damage, mammalian cells adopt checkpoint regulation, by phosphorylation and stabilization of p53, to delay cell cycle progression. However, most cancer cells that lack functional p53 retain an unknown checkpoint mechanism(s) by which cells are arrested at the G2/M phase. Here we demonstrate that a human homolog of Cds1/Rad53 kinase (hCds1) is rapidly phosphorylated and activated in response to DNA damage not only in normal cells but in cancer cells lacking functional p53. A survey of various cancer cell lines revealed that the expression level of hCds1 mRNA is inversely related to the presence of functional p53. In addition, transfection of normal human fibroblasts with SV40 T antigen or human papilloma viruses E6 or E7 causes a marked induction of hCds1 mRNA, and the introduction of functional p53 into SV40 T antigen- and E6-, but not E7-, transfected cells decreases the hCds1 level, suggesting that p53 negatively regulates the expression of hCds1. In cells without functional ataxia telangiectasia mutated (ATM) protein, phosphorylation and activation of hCds1 were observed in response to DNA damage induced by UV but not by ionizing irradiation. These results suggest that hCds1 is activated through an ATM-dependent as well as -independent pathway and that it may complement the function of p53 in DNA damage checkpoints in mammalian cells.
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Tumour suppressor genes, whose usual function seems to be controlling normal cell proliferation, have been implicated in many inherited and sporadic forms of malignancies Much evidence supports the concept of tumour formation by loss-of-function mutations in suppressor genes, as predicted by the two-hit model of Knudson and DeMars. The suppressor gene, p53, is affected in such a manner by numerous mutations, which occur in a variety of human tumours. These mutations usually represent the loss of one allele and the substitution of a single base in the other. We have now analysed the p53 gene in a family affected by Li-Fraumeni syndrome, a rare autosomal dominant syndrome characterized by the occurrence of diverse mesenchymal and epithelial neoplasms at multiple sites. In some instances the neoplasms seem to be related to exposure to carcinogens, including ionizing radiation. The Li-Fraumeni family that we studied had noncancerous skin fibroblasts (NSF) with an unusual radiation-resistant phenotype. DNA derived from the NSF cells of four family members, spanning two generations, had the same point mutation in codon 245 (GGC----GAC) of the p53 gene. This mutation leads to substitution of aspartic acid for glycine in one of the regions identified as a frequent target of point mutations in p53. The NSF cell lines with the mutation also retained the normal p53 allele. This inherited p53 mutation may predispose the members of this family to increased susceptibility to cancer.
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Eight Icelandic families with multiple cases of breast cancer, and 17 pairs of sisters diagnosed by the age of 50 were analysed for linkage to markers around BRCA1 on chromosome 17q. The sister-pairs are thought to represent a wider population as compared to the larger high-risk families. Tumours were also analysed for LOH involving BRCA1. In accordance with a proposed tumour-suppressive function of BRCA1, and high prevalence of LOH in 'linked' tumours, the paired sisters' tumours were assayed for double LOH events with common alleles retained. No such pair was observed, and LOH events were seemingly randomly distributed at a 38% frequency. This indicates that most or all pairs are due to other genes than BRCA1 or sporadic involvement. Of the eight high-risk families, only one showed convincing evidence of 17q-linkage. Therefore, BRCA1 mutations seem to be a minor explanation of familial risk of breast cancer in Iceland.
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Studies on Icelandic breast cancer families have shown that most of them segregate a 999del5 BRCA2 mutation. Here, we report the frequency of the 999del5 BRCA2 mutation in an Icelandic control population and four different groups of cancer patients diagnosed with (a) breast cancer; (b) ovarian cancer; (c) prostate cancer (patients younger than 65 years); and (d) other cancer types. The proportions of individuals carrying the mutation were 0.4% in the control population and in the patient groups 8.5%, 7.9%, 2.7%, and 1.0%, respectively. Our results indicate that BRCA2 confers a very high risk of breast cancer and is responsible for a substantial fraction of breast and ovarian cancer in Iceland, but only a small proportion of other cancers.
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In response to DNA damage and replication blocks, cells prevent cell cycle progression through the control of critical cell cycle regulators. We identified Chk2, the mammalian homolog of theSaccharomyces cerevisiae Rad53 and Schizosaccharomyces pombe Cds1 protein kinases required for the DNA damage and replication checkpoints. Chk2 was rapidly phosphorylated and activated in response to replication blocks and DNA damage; the response to DNA damage occurred in an ataxia telangiectasia mutated (ATM)–dependent manner. In vitro, Chk2 phosphorylated Cdc25C on serine-216, a site known to be involved in negative regulation of Cdc25C. This is the same site phosphorylated by the protein kinase Chk1, which suggests that, in response to DNA damage and DNA replicational stress, Chk1 and Chk2 may phosphorylate Cdc25C to prevent entry into mitosis.
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Upon DNA damage, the amino terminus of p53 is phosphorylated at a number of serine residues including S20, a site that is particularly important in regulating stability and function of the protein. Because no known kinase has been identified that can modify this site, HeLa nuclear extracts were fractionated and S20 phosphorylation was followed. We discovered that a S20 kinase activity copurifies with the human homolog of the Schizosaccharomyces pombe checkpoint kinase, Chk1 (hCHK1). We confirmed that recombinant hCHK1, but not a kinase-defective version of hCHK1, can phosphorylate p53 in vitro at S20. Additional inducible amino- and carboxy-terminal sites in p53 are also phosphorylated by hCHK1, indicating that this is an unusually versatile protein kinase. It is interesting that hCHK1 strongly prefers tetrameric to monomeric p53 in vitro, consistent with our observation that phosphorylation of amino-terminal sites in vivo requires that p53 be oligomeric. Regulation of the levels and activity of hCHK1 in transfected cells is directly correlated with the levels of p53; expression of either a kinase-defective hCHK1 or antisense hCHK1 leads to reduced levels of cotransfected p53, whereas overexpression of wild-type hCHK1 or the kinase domain of hCHK1 results in increased levels of expressed p53 protein. The human homolog of the second S. pombe checkpoint kinase, Cds1 (CHK2/hCds1), phosphorylates tetrameric p53 but not monomeric p53 in vitro at sites similar to those phosphorylated by hCHK1 kinase, suggesting that both checkpoint kinases can play roles in regulating p53 after DNA damage.
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Chk2 is a protein kinase that is activated in response to DNA damage and may regulate cell cycle arrest. We generated Chk2-deficient mouse cells by gene targeting. Chk2−/− embryonic stem cells failed to maintain γ-irradiation–induced arrest in the G2 phase of the cell cycle. Chk2−/−thymocytes were resistant to DNA damage–induced apoptosis. Chk2−/− cells were defective for p53 stabilization and for induction of p53-dependent transcripts such as p21 in response to γ irradiation. Reintroduction of the Chk2 gene restored p53-dependent transcription in response to γ irradiation. Chk2 directly phosphorylated p53 on serine 20, which is known to interfere with Mdm2 binding. This provides a mechanism for increased stability of p53 by prevention of ubiquitination in response to DNA damage.
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Bell et al. ([1][1]) reported mutations in the hCHK2 gene in families with classical Li-Fraumeni syndrome (LFS) and in Li-Fraumeni–like (LFL) families. We have screened genomic DNA for mutations in this gene in one individual from each of 11 LFS families and 25 LFL families, using primers designed
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The hCHK2 gene encodes the human homolog of the yeast Cds1 and Rad53 G2 checkpoint kinases, whose activation in response to DNA damage prevents cellular entry into mitosis. Here, it is shown that heterozygous germ line mutations in hCHK2occur in Li-Fraumeni syndrome, a highly penetrant familial cancer phenotype usually associated with inherited mutations in theTP53 gene. These observations suggest that hCHK2is a tumor suppressor gene conferring predisposition to sarcoma, breast cancer, and brain tumors, and they also provide a link between the central role of p53 inactivation in human cancer and the well-defined G2 checkpoint in yeast.
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Familial cancer syndromes have helped to define the role of tumor suppressor genes in the development of cancer. The dominantly inherited Li-Fraumeni syndrome (LFS) is of particular interest because of the diversity of childhood and adult tumors that occur in affected individuals. The rarity and high mortality of LFS precluded formal linkage analysis. The alternative approach was to select the most plausible candidate gene. The tumor suppressor gene, p53, was studied because of previous indications that this gene is inactivated in the sporadic (nonfamilial) forms of most cancers that are associated with LFS. Germ line p53 mutations have been detected in all five LFS families analyzed. These mutations do not produce amounts of mutant p53 protein expected to exert a trans-dominant loss of function effect on wild-type p53 protein. The frequency of germ line p53 mutations can now be examined in additional families with LFS, and in other cancer patients and families with clinical features that might be attributed to the mutation.