Metformin is under evaluation as a potential anticancer agent. Expression of total and phospho(Thr172)-adenosine monophosphate-activated kinase-α (AMPKα and pAMPKα(Thr172) respectively), a main metformin target, was examined in radiotherapy treated breast cancers and metformin's ability to modulate Trx system expression and breast cancer radiosensitivity evaluated in vitro.AMPKα and pAMPKα(Thr172) expression was assessed using a discovery (n=166) and validation cohort (n=609). Metformin's role in regulating radioresponse, and Trx family expression, was examined via clonogenic assays and Western blots. Intracellular reactive oxygen species (ROS) levels, cell cycle progression and apoptosis were assessed by flow cytometry.High AMPKα expression associated with improved local recurrence-free (P=0.019), relapse-free (P=0.016) and breast cancer-specific survival (P=0.000065) and was, from multivariate analysis, an independent prognostic factor from the discovery cohort. From the validation cases AMPKα expression associated with relapse-free and breast cancer-specific survival in luminal breast cancers. Metformin substantially increased radiosensitivity, intracellular ROS levels and reduced Trx expression, in luminal breast cancer cells, but had little effect on basal phenotype cells.In conclusion, high AMPKα expression associates with improved prognosis, especially in luminal breast cancer. Metformin preferentially radiosensitises luminal breast cancer cells, potentially due to alterations to intracellular ROS levels via modulation of Trx family protein expression.

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BackgroundAtaxia-telangiectasia mutated (ATM), ataxia-telangiectasia mutated and rad3 related (ATR) and DNA-dependent protein kinase catalytic sub-unit (DNA-PKcs) play critical roles in DNA damage response (DDR) by linking DNA damage sensing to DDR effectors that regulate cell cycle progression and DNA repair. Our objective was to evaluate if ATM, ATR and DNA-PKcs expressions could predict response to therapy and clinical outcome in epithelial ovarian cancers.MethodsWe investigated ATM, ATR, and DNA-PKcs expressions in ovarian epithelial cancers [protein expression (n = 194 patients), mRNA expression (n = 156 patients)] and correlated to clinicopathological outcomes as well as expression of X-ray repair cross-complementing protein 1 (XRCC1), cell division cycle-45 (CDC45), cyclin-dependent kinase 1(CDK1) and Ki-67 in tumours.ResultsHigh ATM protein expression was associated with serous cystadenocarcinomas (p = 0.021) and platinum resistance (p = 0.017). High DNA-PKcs protein expression was associated with serous cystadenocarcinomas (p = 0.006) and advanced stage tumours (p = 0.018). High ATM protein (p = 0.001), high ATM mRNA (p = 0.018), high DNA-PKcs protein (p = 0.002), high DNA-PKcs mRNA (p = 0.044) and high ATR protein (p = 0.001) expressions are correlated with poor ovarian cancer specific survival (OCSS). In multivariate Cox model, high DNA-PKcs (p = 0.006) and high ATR (p = 0.043) protein expressions remain independently associated with poor OCSS.ConclusionsATM, ATR and DNA-PKcs expressions may have prognostic and predictive significances in epithelial ovarian cancer.General significanceThe data presented here provides evidence that ATM, ATR and DNA-PKcs involved in DDR are not only promising biomarkers but are also rational targets for personalized therapy in ovarian cancer.

Aims: Radiotherapy is an important treatment for many types of cancer, but a minority of patients suffer long-term side-effects of treatment. Multiple lines of evidence suggest a role for circadian rhythm in the development of radiotherapy late side-effects. Materials and methods: We carried out a study to examine the effect of radiotherapy timing in two breast cancer patient cohorts. The retrospective LeND cohort comprised 535 patients scored for late effects using the Late Effects of Normal Tissue-Subjective Objective Management Analytical (LENT-SOMA) scale. Acute effects were assessed prospectively in 343 patients from the REQUITE study using the CTCAE v4 scales. Genotyping was carried out for candidate circadian rhythm variants. Results: In the LeND cohort, patients who had radiotherapy in the morning had a significantly increased incidence of late toxicity in univariate (P ¼ 0.03) and multivariate analysis (P ¼ 0.01). Acute effects in the REQUITE group were also significantly increased in univariate analysis after morning treatment (P ¼ 0.03) but not on multivariate analysis. Increased late effects in the LeND group receiving morning radiotherapy were associated with carriage of the PER3 variable number tandem repeat 4/4 genotype (P ¼ 6 Â 10 À3) and the NOCT rs131116075 AA genotype (P ¼ 5 Â 10 À3). Conclusion: Our results suggest that it may be possible to reduce toxicity associated with breast cancer radiotherapy by identifying gene variants that affect circadian rhythm and scheduling for appropriate morning or afternoon radiotherapy.

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.