مجال
التميز
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تميز دراسي وبحثي
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البحوث المنشورة
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البحث (1):
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عنوان البحث:
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Targeting
XRCC1 deficiency in breast cancer for personalized therapy
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تاريخ النشر:
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March,
2013
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موجز عن البحث:
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XRCC1
is a key component of DNA base excision repair, single strand break repair,
and backup nonhomologous end-joining pathway. XRCC1 (X-ray repair
cross-complementing gene 1) deficiency promotes genomic instability,
increases cancer risk, and may have clinical application in breast cancer. We
investigated XRCC1 expression in early breast cancers (n = 1,297) and
validated in an independent cohort of estrogen receptor (ER)-α-negative
breast cancers (n = 281). Preclinically, we evaluated XRCC1-deficient and -proficient
Chinese hamster and human cancer cells for synthetic lethality application
using double-strand break (DSB) repair inhibitors [KU55933 (ataxia
telangectasia-mutated; ATM inhibitor) and NU7441 (DNA-PKcs inhibitor)]. In
breast cancer, loss of XRCC1 (16%) was associated with high grade (P <
0.0001), loss of hormone receptors (P < 0.0001), triple-negative (P <
0.0001), and basal-like phenotypes (P = 0.001). Loss of XRCC1 was associated
with a two-fold increase in risk of death (P < 0.0001) and independently
with poor outcome (P < 0.0001). Preclinically, KU55933
[2-(4-Morpholinyl)-6-(1-thianthrenyl)-4H-pyran-4-one] and NU7441
[8-(4-Dibenzothienyl)-2-(4-morpholinyl)-4H-1-benzopyran-4-one] were
synthetically lethal in XRCC1-deficient compared with proficient cells as
evidenced by hypersensitivity to DSB repair inhibitors, accumulation of DNA
DSBs, G2-M cell-cycle arrest, and induction of apoptosis. This is the first
study to show that XRCC1 deficiency in breast cancer results in an aggressive
phenotype and that XRCC1 deficiency could also be exploited for a novel
synthetic lethality application using DSB repair inhibitors. Cancer Res;
73(5); 1621-34. ©2012 AACR.
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البحث (2):
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عنوان البحث:
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Clinicopathological
significance of KU70/KU80, a key DNA damage repair protein in breast cancer
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تاريخ النشر:
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June,2013
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موجز عن البحث:
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Although the role of BRCA1 and the homologous
recombination (HR) pathway in breast cancer (BC) has been extensively
studied, the alternative repair pathway for DNA double-strand breaks (DSBs),
non-homologous end-joining (NHEJ) remains to be defined. Ku proteins bind to
DNA DSB ends and play a key role in NHEJ. In this study we aimed to assess
the expression and biological significance of the KU70/KU80 heterodimer in
the different molecular classes of BC. The expression of KU70/KU80 was
assessed immunohistochemically in a well-characterised and annotated series
of 1302 unselected invasive BC cases with a long-term follow-up together with
25 cases with known BRCA1 mutations. The results were correlated with
clinicopathological parameters, other DNA repair proteins and patient
outcome. The expression of KU70/KU80 protein was further evaluated in various
BC cell lines using western blotting and reverse-phase protein microarray
(RPPA). Nuclear KU70/KU80 expression was correlated with features of poor
prognosis including higher histological grade, lymphovascular invasion,
negative oestrogen receptor expression, basal-like phenotype, P53 and CHK1
positivity. KU70/KU80 was expressed in all BRCA1-associated tumours and
showed an inverse correlation with nuclear BRCA1 protein and aberrant
cytoplasmic RAD51 expression. RPPA confirmed these results and showed higher
expression of KU70/KU80 in BRCA1-deficient cell line compared to
BRCA1-proficient cell line. KU70/KU80 expression showed an association with
disease-free interval; however, it was not an independent predictor of
outcome. As a conclusion, KU70/KU80 may play a role in DNA DSBs repair in
HR-deficient tumours. Further study of other NHEJ markers in sporadic BC is
warranted.
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البحث (3):
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عنوان البحث:
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Targeting BRCA1-BER deficient breast cancer by ATM
or DNA-PKcs blockade either alone or in combination with cisplatin for
personalized therapy.
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تاريخ النشر:
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August 2014
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موجز عن البحث:
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BRCA1, a key factor in homologous recombination (HR)
repair may also regulate base excision repair (BER). Targeting BRCA1-BER
deficient cells by blockade of ATM and DNA-PKcs could be a promising strategy
in breast cancer. We investigated BRCA1, XRCC1 and pol β protein expression
in two cohorts (n = 1602 sporadic and n = 50 germ-line BRCA1 mutated) and
mRNA expression in two cohorts (n = 1952 and n = 249). Artificial neural
network analysis for BRCA1-DNA repair interacting genes was conducted in 249
tumours. Pre-clinically, BRCA1 proficient and deficient cells were DNA repair
expression profiled and evaluated for synthetic lethality using ATM and
DNA-PKcs inhibitors either alone or in combination with cisplatin. In human
tumours, BRCA1 negativity was strongly associated with low XRCC1, and low pol
β at mRNA and protein levels (p < 0.0001). In patients with BRCA1 negative
tumours, low XRCC1 or low pol β expression was significantly associated with
poor survival in univariate and multivariate analysis compared to high XRCC1
or high pol β expressing BRCA1 negative tumours (ps < 0.05).
Pre-clinically, BRCA1 negative cancer cells exhibit low mRNA and low protein
expression of XRCC1 and pol β. BRCA1-BER deficient cells were sensitive to
ATM and DNA-PKcs inhibitor treatment either alone or in combination with
cisplatin and synthetic lethality was evidenced by DNA double strand breaks
accumulation, cell cycle arrest and apoptosis. We conclude that XRCC1 and pol
β expression status in BRCA1 negative tumours may have prognostic
significance. BRCA1-BER deficient cells could be targeted by ATM or DNA-PKcs
inhibitors for personalized therapy.
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البحث ( 4):
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عنوان البحث:
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Genomic and protein expression analysis reveals flap
endonuclease 1 (FEN1) as a key biomarker in breast and ovarian cancer.
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here
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تاريخ النشر:
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October 2014
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موجز عن البحث:
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FEN1 has key roles in Okazaki fragment maturation
during replication, long patch base excision repair, rescue of stalled
replication forks, maintenance of telomere stability and apoptosis. FEN1 may
be dysregulated in breast and ovarian cancers and have clinicopathological
significance in patients. We comprehensively investigated FEN1 mRNA
expression in multiple cohorts of breast cancer [training set (128), test set
(249), external validation (1952)]. FEN1 protein expression was evaluated in
568 oestrogen receptor (ER) negative breast cancers, 894 ER positive breast
cancers and 156 ovarian epithelial cancers. FEN1 mRNA overexpression was
highly significantly associated with high grade (p = 4.89 × 10(-57)), high
mitotic index (p = 5.25 × 10(-28)), pleomorphism (p = 6.31 × 10(-19)), ER
negative (p = 9.02 × 10(-35)), PR negative (p = 9.24 × 10(-24)), triple
negative phenotype (p = 6.67 × 10(-21)), PAM50.Her2 (p = 5.19 × 10(-13)),
PAM50. Basal (p = 2.7 × 10(-41)), PAM50.LumB (p = 1.56 × 10(-26)),
integrative molecular cluster 1 (intClust.1) (p = 7.47 × 10(-12)), intClust.5
(p = 4.05 × 10(-12)) and intClust. 10 (p = 7.59 × 10(-38)) breast cancers.
FEN1 mRNA overexpression is associated with poor breast cancer specific
survival in univariate (p = 4.4 × 10(-16)) and multivariate analysis (p =
9.19 × 10(-7)). At the protein level, in ER positive tumours, FEN1
overexpression remains significantly linked to high grade, high mitotic index
and pleomorphism (ps < 0.01). In ER negative tumours, high FEN1 is
significantly associated with pleomorphism, tumour type, lymphovascular
invasion, triple negative phenotype, EGFR and HER2 expression (ps < 0.05).
In ER positive as well as in ER negative tumours, FEN1 protein overexpression
is associated with poor survival in univariate and multivariate analysis (ps
< 0.01). In ovarian epithelial cancers, similarly, FEN1 overexpression is
associated with high grade, high stage and poor survival (ps < 0.05). We
conclude that FEN1 is a promising biomarker in breast and ovarian epithelial
cancer.
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البحث (5):
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عنوان البحث:
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Single-strand selective monofunctional uracil-DNA
glycosylase (SMUG1) deficiency is linked to aggressive breast cancer and
predicts response to adjuvant therapy.
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here
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تاريخ النشر:
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Dec 2013
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موجز عن البحث:
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Uracil in DNA is an important cause of mutagenesis.
SMUG1 is a uracil-DNA glycosylase that removes uracil through base excision
repair. SMUG1 also processes radiation-induced oxidative base damage as well
as 5-fluorouracil incorporated into DNA during chemotherapy. We investigated
SMUG1 mRNA expression in 249 primary breast cancers. SMUG1 protein expression
was investigated in 1,165 breast tumours randomised into two cohorts
[training set (n = 583) and test set (n = 582)]. SMUG1 and chemotherapy
response was also investigated in a series of 315 ER-negative tumours (n =
315). For mechanistic insights, SMUG1 was correlated to biomarkers of
aggressive phenotype, DNA repair, cell cycle and apoptosis. Low SMUG1 mRNA
expression was associated with adverse disease specific survival (p = 0.008)
and disease-free survival (p = 0.008). Low SMUG1 protein expression (25 %)
was associated with high histological grade (p < 0.0001), high mitotic
index (p < 0.0001), pleomorphism (p < 0.0001), glandular
de-differentiation (p = 0.0001), absence of hormonal receptors (ER-/PgR-/AR)
(p < 0.0001), presence of basal-like (p < 0.0001) and triple-negative
phenotypes (p < 0.0001). Low SMUG1 protein expression was associated with
loss of BRCA1 (p < 0.0001), ATM (p < 0.0001) and XRCC1 (p < 0.0001).
Low p27 (p < 0.0001), low p21 (p = 0.023), mutant p53 (p = 0.037), low MDM2
(p < 0.0001), low MDM4 (p = 0.004), low Bcl-2 (p = 0.001), low Bax (p =
0.003) and high MIB1 (p < 0.0001) were likely in low SMUG1 tumours. Low
SMUG1 protein expression was associated with poor prognosis in univariate (p
< 0.001) and multivariate analysis (p < 0.01). In ER+ cohort that
received adjuvant endocrine therapy, low SMUG1 protein expression remains
associated with poor survival (p < 0.01). In ER- cohort that received
adjuvant chemotherapy, low SMUG1 protein expression is associated with improved
survival (p = 0.043). Our study suggests that low SMUG1 expression may
correlate to adverse clinicopathological features and predict response to
adjuvant therapy in breast cancer.
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البحث (6):
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عنوان البحث:
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Ataxia telangiectasia mutated and Rad3 related (ATR)
protein kinase inhibition is synthetically lethal in XRCC1 deficient ovarian
cancer cells.
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تاريخ النشر:
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Feb 2013
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موجز عن البحث:
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INTRODUCTION: Ataxia telangiectasia mutated and Rad3
Related (ATR) protein kinase is a key sensor of single-stranded DNA
associated with stalled replication forks and repair intermediates generated
during DNA repair. XRCC1 is a critical enzyme in single strand break repair
and base excision repair. XRCC1-LIG3 complex is also an important contributor
to the ligation step of the nucleotide excision repair response.
METHODS: In the current study, we investigated
synthetic lethality in XRCC1 deficient and XRCC1 proficient Chinese Hamster
ovary (CHO) and human ovarian cancer cells using ATR inhibitors (NU6027). In
addition, we also investigated the ability of ATR inhibitors to potentiate
cisplatin cytotoxicity in XRCC1 deficient and XRCC1 proficient CHO and human
cancer cells. Clonogenic assays, alkaline COMET assays, γH2AX
immunocytochemistry, FACS for cell cycle as well as FITC-annexin V flow
cytometric analysis were performed.
RESULTS: ATR inhibition is synthetically lethal in
XRCC1 deficient cells as evidenced by increased cytotoxicity, accumulation of
double strand DNA breaks, G2/M cell cycle arrest and increased apoptosis.
Compared to cisplatin alone, combination of cisplatin and ATR inhibitor
results in enhanced cytotoxicity in XRCC1 deficient cells compared to XRCC1
proficient cells.
CONCLUSIONS: Our data provides evidence that ATR
inhibition is suitable for synthetic lethality application and cisplatin
chemopotentiation in XRCC1 deficient ovarian cancer cells.
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البحث (7):
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عنوان البحث:
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DNA polymerase β deficiency is linked to aggressive
breast cancer: a comprehensive analysis of gene copy number, mRNA and protein
expression in multiple cohorts.
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here
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تاريخ النشر:
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Jan 2014
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موجز عن البحث:
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Short arm of
chromosome 8 is a hot spot for chromosomal breaks, losses and amplifications
in breast cancer. Although such genetic changes may have phenotypic
consequences, the identity of candidate gene(s) remains to be clearly
defined. Pol β gene is localized to chromosome 8p12-p11 and encodes a key DNA
base excision repair protein. Pol β may be a tumour suppressor and involved
in breast cancer pathogenesis. We conducted the first and the largest study
to comprehensively evaluate pol β in breast cancer. We investigated pol β
gene copy number changes in two cohorts (n = 128 &n = 1952), pol β mRNA
expression in two cohorts (n = 249 &n = 1952) and pol β protein
expression in two cohorts (n = 1406 &n = 252). Artificial neural network
analysis for pol β interacting genes was performed in 249 tumours. For
mechanistic insights, pol β gene copy number changes, mRNA and protein levels
were investigated together in 128 tumours and validated in 1952 tumours. Low
pol β mRNA expression as well as low pol β protein expression was associated
high grade, lymph node positivity, pleomorphism, triple negative, basal-like
phenotypes and poor survival (ps < 0.001). In oestrogen receptor (ER)
positive sub-group that received tamoxifen, low pol β protein remains associated
with aggressive phenotype and poor survival (ps < 0.001). Artificial
neural network analysis revealed ER as a top pol β interacting gene.
Mechanistically, there was strong positive correlation between pol β gene
copy number changes and pol β mRNA expression (p < 0.0000001) and between
pol β mRNA and pol β protein expression (p < 0.0000001). This is the first
study to provide evidence that pol β deficiency is linked to aggressive
breast cancer and may have prognostic and predictive significance in patients.
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البحث (8):
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عنوان البحث:
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SUMOylation
proteins in breast cancer.
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تاريخ النشر:
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March 2014
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موجز عن البحث:
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Small
Ubiquitin-like Modifier proteins (or SUMO) modify the function of protein
substrates involved in various cellular processes including DNA damage
response (DDR). It is becoming apparent that dysregulated SUMO contribute to
carcinogenesis by affecting post-transcriptional modification of key
proteins. It is hypothesised that SUMO contributes to the aggressive nature
of breast cancer particularly those associated with features similar to
breast carcinoma arising in patients with BRCA1 germline mutations. This
study aims to assess the clinical and biological significance of three
members of SUMO in a well-characterised annotated series of BC with emphasis
on DDR. The study cohort comprised primary operable invasive BC including
tumours from patients with known BRCA1 germline mutations. SUMO proteins
PIAS1, PIAS4 and UBC9 were assessed using immunohistochemistry utilising
tissue microarray technology. Additionally, their expression was assessed
using reverse phase protein microarray utilising different cell lines. PIAS1
and UBC9 showed cytoplasmic and/or nuclear expression while PIAS4 was
detected only in the nuclei. There was a correlation between subcellular
localisation and expression of the nuclear transport protein KPNA2. Tumours
showing positive nuclear/negative cytoplasmic expression of SUMO featured
good prognostic characteristics including lower histologic grade and had a
good outcome. Strong correlation with DDR-related proteins including BRCA1,
Rad51, ATM, CHK1, DNA-PK and KU70/KU80 was observed. Correlation with ER and
BRCA1 was confirmed using RPPA on cell lines. SUMO proteins seem to play
important role in BC. Not only expression but also subcellular location is
associated with BC phenotype.
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المؤتمرات العلمية:
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المؤتمر (1):
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عنوان المؤتمر:
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National
Cancer Research Institute
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تاريخ الإنعقاد:
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3-6 November 2013
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مكان
الإنعقاد:
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Liverpool,
UK
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طبيعة المشاركة:
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Poster
presentation
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عنوان المشاركة:
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A
novel synthetic lethality application targeting ATM and DNA-PKcs in BRCA1
deficient cancer cells
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ملخص المشاركة:
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Background/Germ-line mutation in BRCA1,
a key player in Homologous recombination (HR), predispose to hereditary
breast and ovarian cancers. BRCA1 may also transcriptionally regulate
expression of genes involved in DNA base excision repair (BER). Our
hypothesis is that BRCA1 deficient cells are BER deficient and may be
suitable for synthetic lethality (SL) targeting using small molecule
inhibitors of ATM or DNA-PKcs that are involved in DSB signalling.
Method/We profiled BRCA1 deficient
cells (MDA-MB-436 & BRCA1-/- HeLa SilenciX® cells) as well as BRCA1
proficient cells (MCF-7 & BRCA1+/+ HeLa SilenciX® cells) for BER (APE1,
XRCC1, SMUG1 and LIG3) gene (RT-PCR) and protein expression (western blot).
We tested for SL using ATM (KU55933) or DNA-PKcs (NU7441) inhibitors either
alone or in combination with cisplatin. MTS assay, Clonogenic assay, γH2AX
immunocytochemistry, FACS and Annexin V analyses were performed. In 1200
human breast cancers, we evaluated BRCA1, ATM, DNA-PKcs, APE1, POLβ, XRCC1 and
SMUG1 protein expression by immunohistochemistry.
Results/BRCA1-/- cells are
deficient in APE1, XRCC1, SMUG1 and LIG3 at the mRNA and protein level
compared to BRCA1+/+ cells. ATM or DNA-PKcs inhibitors are
synthetically lethal in BRCA1-/- cells as evidenced by
accumulation of DNA double-strand breaks, G2/M cell cycle arrest (for ATM
inhibitor) or G1 arrest (DNA-PKcs inhibitor) and induction of apoptosis.
Cytotoxicity potentiation was also observed in BRCA1-/- cells
treated with cisplatin and ATM or DNA-PKcs inhibitor. In human breast
cancers, low BRCA1 is associated with low ATM, DNA-PKcs, APE1, POLβ, XRCC1
and SMUG1 (ps<0.0001). Low ATM or DNA-PKcs is linked to high grade, high
mitotic index, ER-/PR-/AR- (ps<0.01) and poor survival (p<0.0001). In
multivariate analysis, low ATM remains independently associated with poor
survival (p=002).
Conclusion/ATM or DNA-PKcs inhibition
is synthetically lethal in BRCA1-/- cells. ATM and DNA-PKcs
expression may have prognostic and predictive significance in human breast
cancer.
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المؤتمر (2):
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عنوان المؤتمر:
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National
Cancer Research Institute
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تاريخ الإنعقاد:
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3-6 November 2013
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مكان
الإنعقاد:
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Liverpool,
UK
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طبيعة المشاركة:
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Poster
presentation
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عنوان المشاركة:
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Genomic
and protein expression analysis reveals flap structure-specific endonuclease (FEN1)
as a key prognostic, predictive and therapeutic target in breast and ovarian
cancer.
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ملخص المشاركة:
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Background/FEN1
has key roles in Okazaki fragment maturation during replication, long patch
base excision repair, rescue of stalled replication forks, maintenance of
telomere stability and apoptosis. We hypothesised that FEN1 may be
dysregulated and have clinicopathological and therapeutic significance in
breast and ovarian cancer.
Method/A
whole-genome data-mining approach was undertaken to investigate FEN1 in
multiple cohorts of breast cancer [training set (128), test set (249),
external validation (1952)]. Artificial neural network analysis, ensemble
classification and cross validation analysis of 47,293 probes was performed
in 249 breast tumours. FEN1 protein expression was evaluated in 568 oestrogen
receptor (ER) negative breast cancers, 894 ER positive breast cancers and 156
ovarian cancers. FEN1 knockdown or blockade by a small molecule inhibitor was
investigated for enhancement of chemotherapy sensitivity. A chemical library
of 391,275 compounds was screened to identify FEN1 inhibitors.
Results/FEN1
mRNA over expression was significantly associated with high grade, high
mitotic index, pleomorphism, triple negative & basal-like phenotype,
resistance to endocrine & chemotherapy, and poor survival (ps<0.0001).
Artificial neural network analysis revealed novel FEN1 interaction genes
involved in DNA repair, replication and cell cycle regulation. FEN1 protein
over expression is significantly linked to aggressive phenotype, therapy
resistance, and poor survival in oestrogen receptor (ER) negative breast
cancers, ER positive breast cancers and ovarian epithelial cancers. In cancer
cell lines, FEN1 depletion or inhibition by a small molecule results in
sensitivity to DNA damaging chemotherapy. High throughput screening has
identified novel FEN1 inhibitors for therapeutic evaluation.
Conclusion/We
conclude that FEN1 is a key biomarker as well as an attractive drug target in
breast and ovarian cancer.
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المؤتمر (3):
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عنوان المؤتمر:
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the 6th
Saudi Scientific International Conference.
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تاريخ الإنعقاد:
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14 October 2012
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مكان
الإنعقاد:
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Brunel, UK
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طبيعة المشاركة:
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Oral
presentation
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عنوان المشاركة:
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Synthetic
lethal targeting of BRCA1 deficient cells by ATM and DNA-PKcs inhibitors for
personalized cancer therapy
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ملخص المشاركة:
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Synthetic
lethality (SL) has provided new opportunities for the development of targeted
therapies which will increase therapeutic efficacy and reduce toxicity. BRCA1
germ-line mutation is one of the significant contributor towards hereditary
breast and ovarian cancers. Cells lacking functional BRCA protein are
deficient in Homologous recombination (HR) pathway which is critical for
double strand breaks (DSBs) repair. Thus, the cells will dependent on the
non-homologous end joining (NHEJ) pathway to repair the DSBs.
We are investigating a novel SL in BRCA1
deficient (MDA-MB-436 human breast cancer cell line and BRCA1 deficient HeLa
SilenciX®) cell lines and proficient (MCF-7 human breast cancer cell line and
BRCA1 proficient HeLa SilenciX®) cell lines using ATM (key proteins in the HR
pathway) inhibitor (KU55933) and DNA-PKcs (key proteins in the NHEJ pathway)
inhibitor (NU7441) as an alternative strategy.
Recently,
it has been found that BRCA1 may transcriptionally regulate expression of DNA
base excision repair (BER) factors such as OGG1, NTH1 and APE1 which is
essential for processing base damage induced by alkylating agents and
radiation. We are examining BER expression profiling in BRCA1 deficient
cancer cell lines.
Cytotoxicity studies data shows BRCA1
deficient cells are hypersensitive to ATM inhibitor (KU55933) and DNA-PKcs
inhibitor (NU7441). Moreover BER profiling using Western blot analysis shows
that BRCA1 deficient cells have reduced expression of BER enzymes (APE1,
SMUG1, UNG, XRCC1, FEN1, POLY ß). Interestingly, octamer binding
transcription factor OCT1 is also down regulated in BRCA1 deficient cells
which may have a role in BRCA1 mediated stimulation of BER enzymes.
We provide the preliminary evidence that
ATM and DNA-PKcs inhibitors could be alternative synthetic lethality target
in BRCA1 deficient breast cancer evidenced by hypersensitivity to DSB repair
inhibitors.
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