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.
Disruption of Cyclin-Dependent Kinase 12 (
) is known to lead to defects in DNA repair and sensitivity to platinum salts and PARP1/2 inhibitors. However,
has also been proposed as an oncogene in ...breast cancer. We therefore aimed to assess the frequency and distribution of CDK12 protein expression by IHC in independent cohorts of breast cancer and correlate this with outcome and genomic status. We found that 21% of primary unselected breast cancers were CDK12 high, and 10.5% were absent, by IHC. CDK12 positivity correlated with HER2 positivity but was not an independent predictor of breast cancer-specific survival taking HER2 status into account; however, absent CDK12 protein expression significantly correlated with a triple-negative phenotype. Interestingly, CDK12 protein absence was associated with reduced expression of a number of DDR proteins including ATR, Ku70/Ku80, PARP1, DNA-PK, and γH2AX, suggesting a novel mechanism of CDK12-associated DDR dysregulation in breast cancer. Our data suggest that diagnostic IHC quantification of CDK12 in breast cancer is feasible, with CDK12 absence possibly signifying defective DDR function. This may have important therapeutic implications, particularly for triple-negative breast cancers.
.
Summary Background Proliferation markers and profiles have been recommended for guiding the choice of systemic treatments for breast cancer. However, the best molecular marker or test to use has not ...yet been identified. We did this study to identify factors that drive proliferation and its associated features in breast cancer and assess their association with clinical outcomes and response to chemotherapy. Methods We applied an artificial neural network-based integrative data mining approach to data from three cohorts of patients with breast cancer (the Nottingham discovery cohort (n=171), Uppsala cohort (n=249), and Molecular Taxonomy of Breast Cancer International Consortium METABRIC cohort; n=1980). We then identified the genes with the most effect on other genes in the resulting interactome map. Sperm-associated antigen 5 ( SPAG5 ) featured prominently in our interactome map of proliferation and we chose to take it forward in our analysis on the basis of its fundamental role in the function and dynamic regulation of mitotic spindles, mitotic progression, and chromosome segregation fidelity. We investigated the clinicopathological relevance of SPAG5 gene copy number aberrations, mRNA transcript expression, and protein expression and analysed the associations of SPAG5 copy number aberrations, transcript expression, and protein expression with breast cancer-specific survival, disease-free survival, distant relapse-free survival, pathological complete response, and residual cancer burden in the Nottingham discovery cohort, Uppsala cohort, METABRIC cohort, a pooled untreated lymph node-negative cohort (n=684), a multicentre combined cohort (n=5439), the Nottingham historical early stage breast cancer cohort (Nottingham-HES; n=1650), Nottingham early stage oestrogen receptor-negative breast cancer adjuvant chemotherapy cohort (Nottingham-oestrogen receptor-negative-ACT; n=697), the Nottingham anthracycline neoadjuvant chemotherapy cohort (Nottingham-NeoACT; n=200), the MD Anderson taxane plus anthracycline-based neoadjuvant chemotherapy cohort (MD Anderson-NeoACT; n=508), and the multicentre phase 2 neoadjuvant clinical trial cohort (phase 2 NeoACT; NCT00455533 ; n=253). Findings In the METABRIC cohort, we detected SPAG5 gene gain or amplification at the Ch17q11.2 locus in 206 (10%) of 1980 patients overall, 46 (19%) of 237 patients with a PAM50-HER2 phenotype, and 87 (18%) of 488 patients with PAM50-LumB phenotype. Copy number aberration leading to SPAG5 gain or amplification and high SPAG5 transcript and SPAG5 protein concentrations were associated with shorter overall breast cancer-specific survival (METABRIC cohort copy number aberration: hazard ratio HR 1·50, 95% CI 1·18–1·92, p=0·00010; METABRIC cohort transcript: 1·68, 1·40–2·01, p<0·0001; and Nottingham-HES-breast cancer cohort protein: 1·68, 1·32–2·12, p<0·0001). In multivariable analysis, high SPAG5 transcript and SPAG5 protein expression were associated with reduced breast cancer-specific survival at 10 years compared with lower concentrations (Uppsala: HR 1·62, 95% CI 1·03–2·53, p=0·036; METABRIC: 1·27, 1·02–1·58, p=0·034; untreated lymph node-negative cohort: 2·34, 1·24–4·42, p=0·0090; and Nottingham-HES: 1·73, 1·23–2·46, p=0·0020). In patients with oestrogen receptor-negative breast cancer with high SPAG5 protein expression, anthracycline-based adjuvant chemotherapy increased breast cancer-specific survival overall compared with that for patients who did not receive chemotherapy (Nottingham-oestrogen receptor-negative-ACT cohort: HR 0·37, 95% CI 0·20–0·60, p=0·0010). Multivariable analysis showed high SPAG5 transcript concentrations to be independently associated with longer distant relapse-free survival after receiving taxane plus anthracycline neoadjuvant chemotherapy (MD Anderson-NeoACT: HR 0·68, 95% CI 0·48–0·97, p=0·031). In multivariable analysis, both high SPAG5 transcript and high SPAG5 protein concentrations were independent predictors for a higher proportion of patients achieving a pathological complete response after combination cytotoxic chemotherapy (MD Anderson-NeoACT: OR 1·71, 95% CI, 1·07–2·74, p=0·024; Nottingham-ACT: 8·75, 2·42–31·62, p=0·0010). Interpretation SPAG5 is a novel amplified gene on Ch17q11.2 in breast cancer. The transcript and protein products of SPAG5 are independent prognostic and predictive biomarkers that might have clinical utility as biomarkers for combination cytotoxic chemotherapy sensitivity, especially in oestrogen receptor-negative breast cancer. Funding Nottingham Hospitals Charity and the John and Lucille van Geest Foundation.
PARP inhibitor maintenance therapy in platinum sensitive sporadic ovarian cancers improves progression free survival. However, biomarker for synthetic lethality in platinum sensitive sporadic disease ...is yet to be defined. ERCC1-XPF heterodimer is a key player in nucleotide excision repair (NER) involved in the repair of platinum induced DNA damage. In the current study, we tested whether ERCC1-XPF deficiency would predict synthetic lethality to the PARP inhibitor Olaparib and platinum sensitivity in ovarian cancers.
ERCC1, XPF and PARP1 protein expression was evaluated in tumors from a cohort of 331 patients treated at Nottingham University Hospitals and correlated to clinicopathological features and survival. Pre-clinically, ERCC1 and XPF was depleted in A2780 (platinum sensitive) and A2780cis (platinum resistant) ovarian cancer cell lines and tested for platinum sensitivity as well as for Olaparib induced synthetic lethality.
Low ERCC1 was significantly associated with improved progression free survival (PFS) in patients with ovarian cancers in univariate (p = 0.001) and multivariate (p = 0.002) analysis. In addition, low ERCC1/low XPF (p = 0.003) or low ERCC1/low PARP1 (p = 0.0001) tumors was also linked to better PFS compared to high ERCC1/high XPF or high ERCC1/high PARP1 tumors. Pre-clinically, ERCC1 or XPF depletion not only increased platinum sensitivity but also increased toxicity to Olaparib therapy. Increased sensitivity was associated with DNA double strand breaks (DSBs) accumulation, cell cycle arrest and increased apoptosis.
The data provide evidence that low ERCC1 is not only a predictor of platinum sensitivity but is also a promising biomarker for Olaparib induced synthetic lethality in ovarian cancers.
•PARP inhibitor therapy improves progression free survival in platinum sensitive ovarian cancers.•Robust biomarker to predict benefit from PARP inhibitor therapy in sporadic tumors is yet to be established.•Here we show that ERCC1-XPF deficiency predicts platinum sensitivity.•ERCC1 or XPF deficient ovarian cancer cells are sensitive to PARP inhibitor Olaparib through synthetic lethality.
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.
•FEN1 has key roles in Okazaki fragment maturation during replication and long patch DNA base excision repair.•FEN1 mRNA and protein expression was comprehensively evaluated in large cohorts of breast cancers.•FEN1 mRNA and protein overexpression is an adverse prognostic and predictive biomarker in breast cancer.•FEN1 was investigated in ovarian cancer and FEN1 protein overexpression has prognostic and predictive significance.
The role of the androgen receptor (AR) in estrogen receptor (ER)-α-positive breast cancer is controversial, constraining implementation of AR-directed therapies. Using a diverse, clinically relevant ...panel of cell-line and patient-derived models, we demonstrate that AR activation, not suppression, exerts potent antitumor activity in multiple disease contexts, including resistance to standard-of-care ER and CDK4/6 inhibitors. Notably, AR agonists combined with standard-of-care agents enhanced therapeutic responses. Mechanistically, agonist activation of AR altered the genomic distribution of ER and essential co-activators (p300, SRC-3), resulting in repression of ER-regulated cell cycle genes and upregulation of AR target genes, including known tumor suppressors. A gene signature of AR activity positively predicted disease survival in multiple clinical ER-positive breast cancer cohorts. These findings provide unambiguous evidence that AR has a tumor suppressor role in ER-positive breast cancer and support AR agonism as the optimal AR-directed treatment strategy, revealing a rational therapeutic opportunity.
Human epidermal growth factor receptor 2 (HER2) plays an important role in breast cancer progression and provides predictive information for response to targeted therapy including trastuzumab ...although this is limited. Downstream pathways, such as PI3K/Akt, are associated with HER2/HER3 heterodimerization promoting survival and proliferation amongst cancer cells. Thus, patient outcome and trastuzumab therapy effectiveness might be further characterised by HER2/HER3 dimerisation and its signalling pathways. HER2/HER3 dimerisation status was assessed, using chromogenic in situ Proximity Ligation Assay, in two breast cancer series: early stage primary breast cancer, including 224 HER2+ patients that were not submitted to trastuzumab, and HER2+ breast cancer where patients were treated with adjuvant trastuzumab (
n
= 143). Levels of biomarkers including PI3K, pAKT, ER, PgR, HER3, BCL2, p53, PTEN and p21 were measured using immunohistochemistry. Levels of HER2/HER3 heterodimers were compared with biomarker expression and patient outcome. An association between high levels of HER2/HER3 dimerisation and absence of hormone receptors, ER and PgR, was observed. We further show for the first time the presence of HER2/HER3 heterodimers and the loss of p21 expression in HER2+ breast cancer predicts a significantly poorer outcome when submitted to adjuvant trastuzumab. Breast cancer patients that reveal high levels of HER2/HER3 dimerisation and loss of p21 are associated with poor survival prognosis in patients with HER2+ breast cancer treated with adjuvant trastuzumab. Further quantification analysis of HER dimer/ligand complexes and downstream signalling pathways will begin to unravel the complex associations with patient outcome and its relationship with sensitivity to targeted treatment.
Oestrogen metabolites can induce oxidative DNA base damage and generate potentially mutagenic apurinic sites (AP sites) in the genomic DNA. If unrepaired, mutagenic AP sites could drive breast cancer ...pathogenesis and aggressive phenotypes. Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key DNA base excision repair (BER) protein and essential for processing AP sites generated either directly by oestrogen metabolites or during BER of oxidative base damage. Our hypothesis is that altered APE1 expression may be associated with aggressive tumour biology and impact upon clinical outcomes in breast cancer. In the current study, we have investigated APE1 protein expression in a large cohort of breast cancers (
n
= 1285) and correlated to clinicopathological features and survival outcomes. Low APE1 protein expression was associated with high histological grade (
p
< 0.000001), high mitotic index (
p
< 0.000001), glandular de-differentiation (
p
< 0.000001), pleomorphism (
p
= 0.003), absence of hormonal receptors (ER−/PgR−/AR−) (
p
< 0.0001) and presence of triple negative phenotype (
p
= 0.001). Low APE1 protein expression was associated with loss of BRCA1, low XRCC1, low FEN1, low SMUG1 and low pol β (
ps
< 0.0001). High MIB1 (
p
= 0.048), bcl-2 negativity (
p
< 0.0001) and low TOP2A (
p
< 0.0001) were likely in low APE1 tumours. In the ER-positive sub-group, specifically, low APE1 remains significantly associated with high histological grade, high mitotic index, glandular de-differentiation (
ps
< 0.00001) and poor breast cancer specific survival (
p
= 0.007). In the ER-positive cohort that received adjuvant endocrine therapy, low APE1 protein expression is associated with poor survival (
p
= 0.006). In multivariate analysis, low APE1 remains independently associated with poor survival in ER-positive tumours (
p
= 0.048). We conclude that low APE1 expression may have prognostic and predictive significance in ER-positive breast cancers.
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.
•BRCA1 may regulate base excision repair (BER).•BRCA1-BER deficient cells may be sensitive to blockade of ATM and DNA-PKcs.•We show that low XRCC1/low pol β have prognostic significance in BRCA1−/− tumours.•ATM or DNA-PKcs inhibitors are synthetically lethal in BRCA1-BER deficient cells.•Synthetic lethality is enhanced in combination with cisplatin.
We have previously provided evidence showing an association between some precursor lesions with low nuclear grade breast carcinomas (LNGBCs). In this study, further immunophenotypic support to our ...proposed route of pathogenesis of LNGBC and their precursor lesions was provided. Precursor lesions including columnar cell lesions, atypical ductal hyperplasia, ductal carcinoma in situ, usual epithelial hyperplasia, and lobular neoplasia were compared with matching "morphologically normal" terminal lobular duct units and matching invasive carcinoma. The epithelial cells in the putative precursor flat epithelial atypia, atypical ductal hyperplasia, lobular neoplasia, ductal carcinoma in situ lesions, and their coexisting LNGBC were negative for basal and myoepithelial markers, but positive for CK19/18/8, estrogen receptor (ER)-alpha, Bcl-2, and cyclin D1. The ER-alpha/ER-beta expression ratio increased during carcinogenesis, as did expression of cyclin D1 and Bcl-2. p53 immunopositivity was found 3% in LNGBC versus 43% in high nuclear grade breast carcinoma (HNGBC), whereas ataxia telangiectasia mutated expression was absent or reduced in 22% of LNGBC versus 53% of HNGBC cases. In summary, our findings support the concept that flat epithelial atypia is the earliest morphologically identifiable nonobligate precursor lesion of LNGBC. These may represent a family of precursor, in situ and invasive neoplastic lesions belonging to the luminal "A" subclass of breast cancer. The balance between ER-alpha and ER-beta expression may be important in driving cyclin D-1 and Bcl-2 expression. Ataxia telangiectasia mutated may be one of the alternative regulatory mechanisms to TP53 mutation or dysfunction in low-grade and high-grade breast carcinoma. Our findings support the concept that progression of LNGBC to HNGBC (basal-like or HER2+) phenotype is an unlikely biologic phenomenon.
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