FEN1 Blockade for Platinum Chemo-Sensitization and Synthetic Lethality in Epithelial Ovarian Cancers.

FEN1 plays critical roles in long patch base excision repair (LP-BER), Okazaki fragment maturation, and rescue of stalled replication forks. In a clinical cohort, FEN1 overexpression is associated with aggressive phenotype and poor progression-free survival after platinum chemotherapy. Pre-clinically, FEN1 is induced upon cisplatin treatment, and nuclear translocation of FEN1 is dependent on physical interaction with importin ß. FEN1 depletion, gene inactivation, or inhibition re-sensitizes platinum-resistant ovarian cancer cells to cisplatin. BRCA2 deficient cells exhibited synthetic lethality upon treatment with a FEN1 inhibitor. FEN1 inhibitor-resistant PEO1R cells were generated, and these reactivated BRCA2 and overexpressed the key repair proteins, POLß and XRCC1. FEN1i treatment was selectively toxic to POLß deficient but not XRCC1 deficient ovarian cancer cells. High throughput screening of 391,275 compounds identified several FEN1 inhibitor hits that are suitable for further drug development. We conclude that FEN1 is a valid target for ovarian cancer therapy.

Catalytically propelled 3D printed colloidal microswimmers.

Synthetic microswimmers are widely employed model systems in the studies of out-of-equilibrium phenomena. Unlike biological microswimmers which naturally occur in various shapes and forms, synthetic microswimmers have so far been limited almost exclusively to spherical shapes. Here, we exploit 3D printing to produce microswimmers with complex shapes in the colloidal size regime. We establish the flexibility of 3D printing by two-photon polymerisation to produce particles smaller than 10 microns with a high-degree of shape complexity. We further demonstrate that 3D printing allows control over the location of the active site through orienting the particles in different directions during printing. We verify that particles behave colloidally by imaging their motion in the passive and active states and by investigating their mean square displacement. In addition, we find that particles exhibit shape-dependant behavior, thereby demonstrating the potential of our method to launch a wide-range of in-depth studies into shape-dependent active motion and behaviour.

Slip Length Dependent Propulsion Speed of Catalytic Colloidal Swimmers near Walls.

Catalytic colloidal swimmers that propel due to self-generated fluid flows exhibit strong affinity for surfaces. Here, we report experimental measurements of a significant dependence of such microswimmers' speed on the nearby substrate material. We find that speeds scale with the solution contact angle θ on the substrate, which relates to the associated hydrodynamic substrate slip length, as V∝(cosθ+1)^{-3/2}. We show that such dependence can be attributed to osmotic coupling between swimmers and substrate. Our work points out that hydrodynamic slip at nearby walls, though often unconsidered, can significantly impact microswimmer self-propulsion.

Increased Non-Homologous End Joining Makes DNA-PK a Promising Target for Therapeutic Intervention in Uveal Melanoma.

Uveal melanoma (UM) is the most common primary intraocular tumour in adults, with a mean survival of six months following metastasis. The survival rates have not improved in over 30 years. This study has shown that sister chromatid exchange (SCE) is low in UM which is likely due to a reduced expression of FANCD2. As FANCD2 can function to suppress non-homologous end joining (NHEJ), this study therefore investigated NHEJ in UM. The activation of the catalytic subunit of the NHEJ pathway protein DNA-dependent protein kinase (DNA-PK) was measured by analysing the foci formation and the ligation efficiency by NHEJ determined using a plasmid-based end-joining assay. Using small-interfering RNA (siRNA) knock-down, and chemical inhibitors of DNA-PK, the survival of primary UM cultures and two cell lines were determined. To assess the homologous recombination capacity in response to the inhibition of DNA-PK, a SCE analysis was performed. In addition, to support the findings, the messenger RNA (mRNA) expression of genes associated with NHEJ was analysed using the Cancer Genome Atlas (TCGA)-UM RNAseq data (n = 79). The NHEJ activity and DNA-PKcs activation was upregulated in UM and the inhibition of DNA-PK selectively induced apoptosis and sensitized to ionising radiation and inter-strand cross-linking agents. The inhibition of the NHEJ protein DNA-PK is lethal to UM, indicating a potentially effective therapeutic option, either alone or as a sensitizer for other treatments.

Genetic Profiling of Primary Orbital Melanoma: An Analysis of 6 Cases with Clinicopathologic Correlation.

PURPOSE: To analyze the genetic profile of 6 cases of primary orbital melanoma with clinicopathologic correlation. DESIGN: Retrospective noninterventional study to analyze the genetic profile of 6 cases of primary orbital melanoma and to correlate the genetic findings with prognosis and clinicopathologic features. Inclusion criteria were patients with primary orbital melanoma with no evidence of primary eyelid skin, conjunctival, uveal, or remote melanoma at extraocular sites. PARTICIPANTS: The study involved 6 primary orbital melanomas from 6 patients. Four patients were exenterated and 2 had incisional biopsies performed. METHODS: Clinical notes and radiologic records were assessed to ascertain clinical tumor behavior. Sections were stained with hematoxylin-eosin and exposed to immunohistochemistry for S100, MelA, HMB45, Sox10, and BAP1. Melanoma DNA was exposed to array comparative genomic hybridization to assess gross chromosomal copy number changes. Point mutation assessment and Sanger sequencing were performed for GNAQ, GNA11, BRAF, NRAS, pTERT, SF3B1, and EIF1AX. MAIN OUTCOME MEASURES: These were the presence of gross chromosomal copy number changes and the presence of mutations in GNAQ, GNA11, BRAF, NRAS, pTERT, SF3B1, and EIF1AX; the presence of metastases and time period between diagnosis and death from melanoma; and correlation between the tumor genetic profile and the clinical behavior of the tumor. RESULTS: One of the 6 cases was clinically associated with oculodermal melanocytosis. Of the 6 patients, 3 died of melanoma metastases and 1 of unrelated causes; 2 remain alive at last review. Three of the 6 cases were histologically associated with a benign precursor lesion. All melanomas expressed S100, MelA, HMB45, and Sox10. One patient showed loss of BAP1 nuclear staining. The most frequent chromosomal gains across the 6 cases, in order of frequency, were 6p, 8q, 17q, 6q, and 20p. The most frequently lost regions were 1p, 9p, 16q, and 17p. One patient showed monsomy 3 and gain of 8q (and showed the BAP1 loss). Mutations were found in GNAQ (1 case), GNA11 (1 case), SF3B1 (2 cases), NRAS (2 cases), and pTERT (2 cases). CONCLUSIONS: The data point to 2 genetic groups for primary orbital conjunctiva melanoma-like and a uveal melanoma-like group. A larger study would help confirm this suggestion.

Developing leadership as a trainee- opportunities, barriers and potential improvements.

The General Medical Council explicitly state that doctors completing training should demonstrate capabilities in leadership and teamwork.1 However, most trainees receive little formal training in leadership. In March 2017, at the Faculty of Medical Leadership and Management (FMLM) Northern Ireland Regional Conference, a workshop on developing leadership skills as a trainee was hosted and the views of doctors in training regarding current opportunities, potential barriers and improvements were sought. In Northern Ireland presently there are a number of opportunities available for trainees to gain experience in leadership - both by learning through observation and learning through experience. These range from informal activities which do not require significant time commitment to focused, immersive leadership experiences such as ADEPT (Achieve Develop Explore Programme for Trainees)2, and the Royal College of Physicians' Chief Registrar scheme.3 Several barriers to developing leadership have been identified, including limited understanding of what constitutes leadership, a lack of senior support and little formal recognition for trainees leading teams. Time pressures, frequently rotating jobs, limited resources and difficulty upscaling can also undermine the sustainability of improvement and other leadership projects. Incorporating awareness of and training in leadership skills, as well as greater engagement with senior leaders and managers, at an early stage in training could promote understanding and encourage trainees. Formalising leadership roles within training posts may improve experience. Deaneries and Trusts can also enable leadership opportunities by facilitating study leave, raising awareness amongst supervisors, and providing career enhancing incentives for interested trainees.

Phenotypic Plasticity in Uveal Melanoma Is Not Restricted to a Tumor Subpopulation and Is Unrelated to Cancer Stem Cell Characteristics.

Purpose: Uveal melanoma (UM) is the most common primary intraocular malignancy in adults and approximately half of those diagnosed will die of metastasis. This study investigates whether UM progression is driven by a subpopulation of stem-like cells, termed "cancer stem cells" (CSCs). Methods: Expression of postulated stem cell markers aldehyde dehydrogenase (ALDH), CD44, and CD133 was analyzed in UM cell lines and primary UM short-term cultures (STCs) established from tumor samples. Additionally, the notion of a "cellular hierarchy" within UM was investigated. Finally, the phenomenon of phenotypic plasticity in response to environmental factors was explored. Results: We demonstrate that expression of ALDH, CD44, and CD133 does not select for a subpopulation of stem-like cells in either UM cell lines or UM STCs. Furthermore, there is an absence of a cellular hierarchy in cell lines and all cells in culture are able to drive tumor progression. Last, we show that established UM cell lines and UM STCs are plastic in nature and switch their phenotype in response to environmental stimuli. Conclusions: We hypothesize that this capacity to undergo phenotypic plasticity may be a consequence of neural crest lineage and renders the exploration of the CSC hypothesis extremely challenging in UM.

Knowledge, attitudes and practices towards yaws and yaws-like skin disease in Ghana.

INTRODUCTION: Yaws is endemic in Ghana. The World Health Organization (WHO) has launched a new global eradication campaign based on total community mass treatment with azithromycin. Achieving high coverage of mass treatment will be fundamental to the success of this new strategy; coverage is dependent, in part, on appropriate community mobilisation. An understanding of community knowledge, attitudes and practices related to yaws in Ghana and other endemic countries will be vital in designing effective community engagement strategies. METHODS: A verbally administered questionnaire was administered to residents in 3 districts in the Eastern region of Ghana where a randomised trial on the treatment of yaws was being conducted. The questionnaire combined both quantitative and qualitative questions covering perceptions of the cause and mechanisms of transmission of yaws-like lesions, the providers from which individuals would seek healthcare for yaws-like lesions, and what factors were important in reaching decisions on where to seek care. Chi-square tests and logistic regression were used to assess relationships between reported knowledge, attitudes and practices, and demographic variables. Thematic analysis of qualitative data was used to identify common themes. RESULTS: A total of 1,162 individuals participated. The majority of individuals (n = 895, 77%) reported that "germs" were the cause of yaws lesions. Overall 13% (n = 161) of respondents believed that the disease was caused by supernatural forces. Participants frequently mentioned lack of personal hygiene, irregular and inefficient bathing, and washing with dirty water as fundamental to both the cause and the prevention of yaws. A majority of individuals reported that they would want to take an antibiotic to prevent the development of yaws if they were asymptomatic (n = 689, 61.2%), but a substantial minority reported they would not want to do so. A majority of individuals (n = 839, 72.7%) reported that if they had a yaws-like skin lesion they would seek care from a doctor or nurse. Both direct and indirect costs of treatment were reported as key factors affecting where participants reported they would seek care. DISCUSSION: This is the first study that has explored community knowledge, attitudes and practices in relation to yaws in any endemic population. The belief that 'germs' are in some way related to disease through a variety of transmission routes including both contact and dirty water are similar to those reported for other skin diseases in Ghana. The prominent role of private healthcare providers is an important finding of this study and suggests engagement with this sector will be important in yaws eradication efforts. Strategies to address the substantial minority of individuals who reported they would not take treatment for yaws if they were currently asymptomatic will be needed to ensure the success of yaws eradication efforts. The data collected will be of value to the Ghana Health Service and also to WHO and other partners, who are currently developing community mobilisation tools to support yaws eradication efforts worldwide.

Late Solitary Extraocular Recurrence From Previously Resected Iris Melanoma.

PURPOSE: To report on cases of late extraocular relapse of previously resected iris melanoma, without concurrent intraocular recurrence. DESIGN: Retrospective case series. METHODS: A retrospective chart review of 4 patients diagnosed with late subconjunctival relapse of previously resected iris melanoma. RESULTS: Three female patients and 1 male patient underwent iris tumor resection and presented to our service with suspicious conjunctival lesions at a median of 22 years later (mean: 21 years). None showed intraocular relapse. Treatment of the conjunctival tumors included excisional biopsy (n = 4), followed by cryotherapy (n = 3) and/or brachytherapy (n = 3). In all cases, histopathology confirmed malignant melanoma, with no intraepithelial component or associated melanosis. Genetic sequencing (n = 3) showed wild-type BRAF and NRAS in all. GNA11 mutation was found in 1 case. On array-based comparative genomic hybridization (n = 3), gain of 6p was found in 2 cases and gain of 8 in 2. Overall, findings were strongly suggestive of a diagnosis of late extraocular relapse from previously resected iris melanoma. In a median of 2.5 years (mean: 7.7 years) from the subconjunctival relapse, no further episodes of intraocular/extraocular recurrence were recorded, and all patients were free from distant metastasis. CONCLUSIONS: Patients undergoing iris melanoma resection are at risk of developing late solitary extraocular relapse even more than 30 years after surgery. In the absence of an intraocular component, diagnosis may be challenging, as tumors mimic a primary conjunctival lesion. Management by excisional biopsy followed by adjuvant therapy was successful, and histopathology and genetic analysis supported a diagnosis of extraocular uveal tumor spread rather than a primary conjunctival tumor.

Teenage motherhood: where you live is also important. A prospective cohort study of 14,000 women.

The United Kingdom has among the highest rates of teenage motherhood (TM) in Western Europe. The relationship to individual social and material disadvantage is well established but the influence of area of residence is unclear. We tested for additional TM risks in deprived areas or in cities. The Northern Ireland Longitudinal Study was used to identify 14,055 nulliparous females (15-18). TM risk was measured using multilevel logistic regression, adjusting for health status, religion, family structure, socio-economic status, rurality and employment-based area deprivation. Most variation in TM was driven by individual, household and socioeconomic factors with the greatest proportion of mothers in low value or social rented accommodation. Living in an area with fewer employment opportunities was associated with elevated TM risk (most vs. least deprived, ORadj =1.98 [1.49, 2.63]), as was urban dwelling (urban vs. intermediate, ORadj =1.42 [1.13, 1.78]). We conclude that area of residence is a significant independent risk factor for TM. Interventions should be targeted towards the most deprived and urban areas and to those in the lowest value housing.

Photodynamic killing of cancer cells by a Platinum(II) complex with cyclometallating ligand.

Photodynamic therapy that uses photosensitizers which only become toxic upon light-irradiation provides a strong alternative to conventional cancer treatment due to its ability to selectively target tumour material without affecting healthy tissue. Transition metal complexes are highly promising PDT agents due to intense visible light absorption, yet the majority are toxic even without light. This study introduces a small, photostable, charge-neutral platinum-based compound, Pt(II) 2,6-dipyrido-4-methyl-benzenechloride, complex 1, as a photosensitizer, which works under visible light. Activation of the new photosensitizer at low concentrations (0.1-1 µM) by comparatively low dose of 405 nm light (3.6 J cm(-2)) causes significant cell death of cervical, colorectal and bladder cancer cell lines, and, importantly, a cisplatin resistant cell line EJ-R. The photo-index of the complex is 8. We demonstrate that complex 1 induces irreversible DNA single strand breaks following irradiation, and that oxygen is essential for the photoinduced action. Neither light, nor compound alone led to cell death. The key advantages of the new drug include a remarkably fast accumulation time (diffusion-controlled, minutes), and photostability. This study demonstrates a highly promising new agent for photodynamic therapy, and attracts attention to photostable metal complexes as viable alternatives to conventional chemotherapeutics, such as cisplatin.

Chk1 phosphorylated at serine345 is a predictor of early local recurrence and radio-resistance in breast cancer.

Radiation-induced DNA damage activates the DNA damage response (DDR). DDR up-regulation may predict radio-resistance and increase the risk of early local recurrence despite radiotherapy in early stage breast cancers. In 1755 early stage breast cancers, DDR signalling [ATM, ATR, total Ckh1, Chk1 phosphorylated at serine(345) (pChk1), Chk2, p53], base excision repair [PARP1, POLß, XRCC1, FEN1, SMUG1], non-homologous end joining (Ku70/Ku80, DNA-PKcs) and homologous recombination [RAD51, BRCA1, γH2AX, BLM, WRN, RECQL5, PTEN] protein expression was correlated to time to early local recurrence. Pre-clinically, radio-sensitization by inhibition of Chk1 activation by ATR inhibitor (VE-821) and inhibition of Chk1 (V158411) were investigated in MDA-MB-231 (p53 mutant) and MCF-7 (p53 wild-type) breast cancer cells. In the whole cohort, 208/1755 patients (11.9%) developed local recurrence of which 126 (61%) developed local recurrence within 5 years of initiation of primary therapy. Of the 20 markers tested, only pChk1 and p53 significantly associated with early local recurrence (p value = 0.015 and 0.010, respectively). When analysed together, high cytoplasmic pChk1-nuclear pChk1 (p = 0.039), high cytoplasmic pChk1-p53 (p = 0.004) and high nuclear pChk1-p53 (p = 0.029) co-expression remain significantly linked to early local recurrence. In multivariate analysis, cytoplasmic pChk1 level independently predicted early local recurrence (p = 0.025). In patients who received adjuvant local radiotherapy (n = 949), p53 (p = 0.014) and high cytoplasmic pChk1-p53 (p = 0.017) remain associated with early local recurrence. Pre-clinically, radio-sensitisation by VE-821 or V158411 was observed in both MCF-7 and MDA-MB-231 cells and was more pronounced in MCF-7 cells. We conclude that pChk1 is a predictive biomarker of radiotherapy resistance and early local recurrence.

Clinicopathological and prognostic significance of RECQL5 helicase expression in breast cancers.

RECQL5 is a member of the RecQ family of DNA helicases and has key roles in homologous recombination, base excision repair, replication and transcription. The clinicopathological significance of RECQL5 expression in breast cancer is unknown. In this study, we have evaluated RECQL5 mRNA expression in 1977 breast cancers, and RECQL5 protein level in 1902 breast cancers [Nottingham Tenovus series (n = 1650) and ER- cohort (n = 252)]. Expression levels were correlated to aggressive phenotypes and survival outcomes. High RECQL5 mRNA expression was significantly associated with high histological grade (P = 0.007), HER2 overexpression (P = 0.032), ER+/HER2-/high proliferation genefu subtype (P < 0.0001), integrative molecular clusters (intClust 1and 9) (P < 0.0001) and poor survival (P < 0.0001). In subgroup analysis, high RECQL5 mRNA level remains significantly associated with poor BCSS in ER+ cohort (P < 0.0001) but not in ER- cohort (P = 0.116). At the protein level, in tumours with low RAD51, high RECQL5 level was significantly associated with high histological grade (P < 0.0001), higher mitotic index (P = 0.008), dedifferentiation (P = 0.025), pleomorphism (P = 0.027) and poor survival (P = 0.003). In subgroup analysis, high RECQL5/low RAD51 remains significantly associated with poor BCSS in ER+ cohort (P = 0.010), but not in ER- cohort (P = 0.628). In multivariate analysis, high RECQL5 mRNA and high RECQL5/low RAD51 nuclear protein coexpression independently influenced survival (P = 0.022) in whole cohort and in the ER+ subgroup. Preclinically, we show that exogenous expression of RECQL5 in MCF10A cells can drive proliferation supporting an oncogenic function for RECQL5 in breast cancer. We conclude that RECQL5 is a promising biomarker in breast cancer.

Transcriptomic profiling of Forkhead box transcription factors in adult glioblastoma multiforme.

BACKGROUND: The Forkhead box transcription factor (FOX) family plays an essential role in embryogenesis, especially during brain development. Our hypothesis is that de-regulation of FOX genes may contribute to aggressive tumor biology and therapy resistance in patients with glioblastoma multiforme (GBM). MATERIALS AND METHODS: Univariate and multivariate analyses were performed to evaluate prognostic significance of transcript levels of 31 FOX genes in a test set of GBM patients (n=191) and validated them in The Cancer Genome Atlas (TCGA) cohort comprising of 508 adult cases of GBM. The predictive significance of key FOX genes was investigated in patients who received chemotherapy or radiotherapy. RESULTS: Low FOXA2 mRNA, low FOXN2 mRNA, low FOXN3 mRNA and high FOXG1 mRNA were associated with poor survival in the test and TCGA validation cohorts. In multivariate analysis, low FOXA2 mRNA, low FOXN2 mRNA, low FOXN3 mRNA and high FOXG1 mRNA remained independently associated with poor survival in the test and TCGA validation cohorts. In patients who received chemotherapy or radiotherapy, low FOXA2 mRNA, low FOXN2 mRNA and high FOXG1 mRNA correlated with adverse outcomes in the TCGA validation cohort. CONCLUSION: To our knowledge, our data provide the first comprehensive clinical evidence that FOXA2, FOXN2, FOXN3 and FOXG1 are promising biomarkers of GBM and warrant further investigation.

DNA Repair Endonucleases: Physiological Roles and Potential as Drug Targets.

Genomic DNA is constantly exposed to endogenous and exogenous damaging agents. To overcome these damaging effects and maintain genomic stability, cells have robust coping mechanisms in place, including repair of the damaged DNA. There are a number of DNA repair pathways available to cells dependent on the type of damage induced. The removal of damaged DNA is essential to allow successful repair. Removal of DNA strands is achieved by nucleases. Exonucleases are those that progressively cut from DNA ends, and endonucleases make single incisions within strands of DNA. This review focuses on the group of endonucleases involved in DNA repair pathways, their mechanistic functions, roles in cancer development, and how targeting these enzymes is proving to be an exciting new strategy for personalized therapy in cancer.

Transcriptomic and Protein Expression Analysis Reveals Clinicopathological Significance of Bloom Syndrome Helicase (BLM) in Breast Cancer.

Bloom syndrome helicase (BLM) has key roles in homologous recombination repair, telomere maintenance, and DNA replication. Germ-line mutations in the BLM gene causes Bloom syndrome, a rare disorder characterized by premature aging and predisposition to multiple cancers, including breast cancer. The clinicopathologic significance of BLM in sporadic breast cancers is unknown. We investigated BLM mRNA expression in the Molecular Taxonomy of Breast Cancer International Consortium cohort (n = 1,950) and validated in an external dataset of 2,413 tumors. BLM protein level was evaluated in the Nottingham Tenovus series comprising 1,650 breast tumors. BLM mRNA overexpression was significantly associated with high histologic grade, larger tumor size, estrogen receptor-negative (ER(-)), progesterone receptor-negative (PR(-)), and triple-negative phenotypes (ps < 0.0001). BLM mRNA overexpression was also linked to aggressive molecular phenotypes, including PAM50.Her2 (P < 0.0001), PAM50.Basal (P < 0.0001), and PAM50.LumB (P < 0.0001) and Genufu subtype (ER(+)/Her2(-)/high proliferation; P < 0.0001). PAM50.LumA tumors and Genufu subtype (ER(+)/Her2(-)/low proliferation) were more likely to express low levels of BLM mRNA (ps < 0.0001). Integrative molecular clusters (intClust) intClust.1 (P < 0.0001), intClust.5 (P < 0.0001), intClust.9 (P < 0.0001), and intClust.10 (P < 0.0001) were also more likely in tumors with high BLM mRNA expression. BLM mRNA overexpression was associated with poor breast cancer-specific survival (BCSS; ps < 0.000001). At the protein level, altered subcellular localization with high cytoplasmic BLM and low nuclear BLM was linked to aggressive phenotypes. In multivariate analysis, BLM mRNA and BLM protein levels independently influenced BCSS. This is the first and the largest study to provide evidence that BLM is a promising biomarker in breast cancer.

Targeting BRCA1-BER deficient breast cancer by ATM or DNA-PKcs blockade either alone or in combination with cisplatin for personalized therapy.

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 ß 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 ß at mRNA and protein levels (p < 0.0001). In patients with BRCA1 negative tumours, low XRCC1 or low pol ß expression was significantly associated with poor survival in univariate and multivariate analysis compared to high XRCC1 or high pol ß expressing BRCA1 negative tumours (ps < 0.05). Pre-clinically, BRCA1 negative cancer cells exhibit low mRNA and low protein expression of XRCC1 and pol ß. 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 ß 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.

Untangling the ATR-CHEK1 network for prognostication, prediction and therapeutic target validation in breast cancer.

ATR-CHEK1 signalling is critical for genomic stability. ATR-CHEK1 signalling may be deregulated in breast cancer and have prognostic, predictive and therapeutic significance. We investigated ATR, CHEK1 and phosphorylated CHEK1 (Ser345) protein (pCHEK1) levels in 1712 breast cancers. ATR and CHEK1 mRNA expression was evaluated in 1950 breast cancers. Pre-clinically, biological consequences of ATR gene knock down or ATR inhibition by the small molecule inhibitor (VE-821) were investigated in MCF7 and MDA-MB-231 breast cancer cell lines and in non-tumorigenic breast epithelial cells (MCF10A). High ATR and high cytoplasmic pCHEK1 levels were significantly associated with higher tumour stage, higher mitotic index, pleomorphism and lymphovascular invasion. In univariate analyses, high ATR and high cytoplasmic pCHEK1 levels were associated with poor breast cancer specific survival (BCSS). In multivariate analysis, high ATR level remains an independent predictor of adverse outcome. At the mRNA level, high CHEK1 remains associated with aggressive phenotypes including lymph node positivity, high grade, Her-2 overexpression, triple negative, aggressive molecular phenotypes and adverse BCSS. Pre-clinically, CHEK1 phosphorylation at serine(345) following replication stress was impaired in ATR knock down and in VE-821 treated breast cancer cells. Doxycycline inducible knockdown of ATR suppressed growth, which was restored when ATR was re-expressed. Similarly, VE-821 treatment resulted in a dose dependent suppression of cancer cell growth and survival (MCF7 and MDA-MB-231) but was less toxic in non-tumorigenic breast epithelial cells (MCF10A). We provide evidence that ATR and CHEK1 are promising biomarkers and rational drug targets for personalized therapy in breast cancer.

Clinicopathological significance of ATM-Chk2 expression in sporadic breast cancers: a comprehensive analysis in large cohorts.

ATM-Chk2 network is critical for genomic stability, and its deregulation may influence breast cancer pathogenesis. We investigated ATM and Chk2 protein levels in two cohorts [cohort 1 (n = 1650) and cohort 2 (n = 252)]. ATM and Chk2 mRNA expression was evaluated in the Molecular Taxonomy of Breast Cancer International Consortium cohort (n = 1950). Low nuclear ATM protein level was significantly associated with aggressive breast cancer including larger tumors, higher tumor grade, higher mitotic index, pleomorphism, tumor type, lymphovascular invasion, estrogen receptor (ER)-, PR -, AR -, triple-negative, and basal-like phenotypes (Ps < .05). Breast cancer 1, early onset negative, low XRCC1, low SMUG1, high FEN1, high MIB1, p53 mutants, low MDM2, low Bcl-2, low p21, low Bax, high CDK1, and low Chk2 were also more frequent in tumors with low nuclear ATM level (Ps < .05). Low ATM protein level was significantly associated with poor survival including in patients with ER-negative tumors who received adjuvant anthracycline or cyclophosphamide, methotrexate, and 5-fluorouracil-based adjuvant chemotherapy (Ps < .05). Low nuclear Chk2 protein was likely in ER -/PR -/AR -; HER-2 positive; breast cancer 1, early onset negative; low XRCC1; low SMUG1; low APE1; low polß; low DNA-PKcs; low ATM; low Bcl-2; and low TOPO2A tumors (P < .05). In patients with ER + tumors who received endocrine therapy or ER-negative tumors who received chemotherapy, nuclear Chk2 levels did not significantly influence survival. In p53 mutant tumors, low ATM (P < .000001) or high Chk2 (P < .01) was associated with poor survival. When investigated together, low-ATM/high-Chk2 tumors have the worst survival (P = .0033). Our data suggest that ATM-Chk2 levels in sporadic breast cancer may have prognostic and predictive significance.

Is there a role for base excision repair in estrogen/estrogen receptor-driven breast cancers?

Estrogen and estrogen metabolite-induced reactive oxygen species generation can promote oxidative DNA base damage. If unrepaired, base damaging lesions could accelerate mutagenesis, leading to a "mutator phenotype" characterized by aggressive behavior in estrogen-estrogen receptor (ER)-driven breast cancer. To test this hypothesis, we investigated 1406 ER(+) early-stage breast cancers with 20 years' long-term clinical follow-up data for DNA polymerase ß (pol ß), flap endonuclease 1 (FEN1), AP endonuclease 1 (APE1), X-ray cross-complementation group 1 protein (XRCC1), single-strand monofunctional uracil glycosylase-1 (SMUG1), poly (ADP-ribose) polymerase 1 (PARP1), ataxia telangiectasia mutated and Rad3 related (ATR), ataxia telangiectasia mutated (ATM), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Chk1, Chk2, p53, breast cancer susceptibility gene 1 (BRCA1), and topoisomerase 2 (TOPO2) expression. Multivariate Cox proportional hazards model was used to calculate a DNA repair prognostic index and correlated to clinicopathological variables and survival outcomes. Key base excision repair (BER) proteins, including XRCC1, APE1, SMUG1, and FEN1, were independently associated with poor breast cancer-specific survival (BCSS) (ps≤0.01). Multivariate Cox model stratified patients into four distinct prognostic sub-groups with worsening BCSS (ps<0.01). In addition, compared with prognostic sub-group 1, sub-groups 2, 3, and 4 manifest increasing tumor size, grade, mitosis, pleomorphism, differentiation, lymphovascular invasion, high Ki67, loss of Bcl-2, luminal B phenotype (ps≤0.01), and poor survival, including in patients who received tamoxifen adjuvant therapy (p<0.00001). Our observation supports the hypothesis that BER-directed stratification could inform appropriate therapies in estrogen-ER-driven breast cancers. Antioxid.