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The rapid cell turnover of the intestinal epithelium is achieved from small numbers of stem cells located in the base of glandular crypts. These stem cells have been variously described as rapidly cycling or quiescent. A functional arrangement of stem cells that reconciles both of these behaviours has so far been difficult to obtain. Alternative explanations for quiescent cells have been that they act as a parallel or reserve population that replace rapidly cycling stem cells periodically or after injury; their exact nature remains unknown. Here we show mouse intestinal quiescent cells to be precursors that are committed to mature into differentiated secretory cells of the Paneth and enteroendocrine lineage. However, crucially we find that after intestinal injury they are capable of extensive proliferation and can give rise to clones comprising the main epithelial cell types. Thus, quiescent cells can be recalled to the stem-cell state. These findings establish quiescent cells as an effective clonogenic reserve and provide a motivation for investigating their role in pathologies such as colorectal cancers and intestinal inflammation. A new method to trace the lineage of slow cycling label-retaining cells (LRCs) in vivo identifies a population of LRCs that have features of committed Paneth cells but still express stem-cell markers such as Lgr5; the slow cycling cells differentiate into Paneth cells without cell division, but after injury can also repopulate the stem-cell niche and contribute to the regeneration of all intestinal lineages. Quiescent cells in intestinal epithelium Whether slowly cycling or quiescent stem cells co-exist with more rapidly cycling stem cells in the intestinal crypt is a matter of intense debate. Using a new method to trace the lineage of slowly cycling label-retaining cells (LRCs) in vivo , Douglas Winton and colleagues have identified a population of LRCs that has features of committed Paneth cells but still expresses stem cell markers such as Lgr5. The slowly cycling cells differentiate into Paneth cells without cell division, but in response to injury, they can also repopulate the stem cell niche and contribute to the regeneration of the all intestinal lineages. This work suggests that quiescent cells do act as a clonogenic reserve that could play a part in the pathology of intestinal cancers and inflammation.

The epidermis is maintained by multiple stem cell populations whose progeny differentiate along diverse, and spatially distinct, lineages. Here we show that the transcription factor Gata6 controls the identity of the previously uncharacterized sebaceous duct (SD) lineage and identify the Gata6 downstream transcription factor network that specifies a lineage switch between sebocytes and SD cells. During wound healing differentiated Gata6 + cells migrate from the SD into the interfollicular epidermis and dedifferentiate, acquiring the ability to undergo long-term self-renewal and differentiate into a much wider range of epidermal lineages than in undamaged tissue. Our data not only demonstrate that the structural and functional complexity of the junctional zone is regulated by Gata6, but also reveal that dedifferentiation is a previously unrecognized property of post-mitotic, terminally differentiated cells that have lost contact with the basement membrane. This resolves the long-standing debate about the contribution of terminally differentiated cells to epidermal wound repair. Donati et al. show that following skin wounding a differentiated Gata6 + cell population resident in the sebaceous duct migrates to the interfollicular epidermis and reattaches to the basal membrane, dedifferentiating into stem cells.

Background Cancer progression is associated with genomic instability and an accumulation of gains and losses of DNA. The growing variety of tools for measuring genomic copy numbers, including various types of array-CGH, SNP arrays and high-throughput sequencing, calls for a coherent framework offering unified and consistent handling of single- and multi-track segmentation problems. In addition, there is a demand for highly computationally efficient segmentation algorithms, due to the emergence of very high density scans of copy number. Results A comprehensive Bioconductor package for copy number analysis is presented. The package offers a unified framework for single sample, multi-sample and multi-track segmentation and is based on statistically sound penalized least squares principles. Conditional on the number of breakpoints, the estimates are optimal in the least squares sense. A novel and computationally highly efficient algorithm is proposed that utilizes vector-based operations in R. Three case studies are presented. Conclusions The R package copynumber is a software suite for segmentation of single- and multi-track copy number data using algorithms based on coherent least squares principles.

Understanding breast cancer genetic risk relies on identifying causal variants and candidate target genes in risk loci identified by genome-wide association studies (GWAS), which remains challenging. Since most loci fall in active gene regulatory regions, we developed a novel approach facilitated by pinpointing the variants with greater regulatory potential in the disease’s tissue of origin. Through genome-wide differential allelic expression (DAE) analysis, using microarray data from 64 normal breast tissue samples, we mapped the variants associated with DAE (daeQTLs). Then, we intersected these with GWAS data to reveal candidate risk regulatory variants and analysed their cis-acting regulatory potential. Finally, we validated our approach by extensive functional analysis of the 5q14.1 breast cancer risk locus. We observed widespread gene expression regulation by cis-acting variants in breast tissue, with 65% of coding and noncoding expressed genes displaying DAE (daeGenes). We identified over 54 K daeQTLs for 6761 (26%) daeGenes, including 385 daeGenes harbouring variants previously associated with BC risk. We found 1431 daeQTLs mapped to 93 different loci in strong linkage disequilibrium with risk-associated variants (risk-daeQTLs), suggesting a link between risk-causing variants and cis-regulation. There were 122 risk-daeQTL with stronger cis-acting potential in active regulatory regions with protein binding evidence. These variants mapped to 41 risk loci, of which 29 had no previous report of target genes and were candidates for regulating the expression levels of 65 genes. As validation, we identified and functionally characterised five candidate causal variants at the 5q14.1 risk locus targeting the ATG10 and ATP6AP1L genes, likely acting via modulation of alternative transcription and transcription factor binding. Our study demonstrates the power of DAE analysis and daeQTL mapping to identify causal regulatory variants and target genes at breast cancer risk loci, including those with complex regulatory landscapes. It additionally provides a genome-wide resource of variants associated with DAE for future functional studies.

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.

BackgroundMediator complex (MED) proteins have a key role in transcriptional regulation, some interacting with the oestrogen receptor (ER). Interrogation of the METABRIC cohort suggested that MED7 may regulate lymphovascular invasion (LVI). Thus MED7 expression was assessed in large breast cancer (BC) cohorts to determine clinicopathological significance.MethodsMED7 gene expression was investigated in the METABRIC cohort (n = 1980) and externally validated using bc-GenExMiner v4.0. Immunohistochemical expression was assessed in the Nottingham primary BC series (n = 1280). Associations with clinicopathological variables and patient outcome were evaluated.ResultsHigh MED7 mRNA and protein expression was associated with good prognostic factors: low grade, smaller tumour size, good NPI, positive hormone receptor status (p < 0.001), and negative LVI (p = 0.04) status. Higher MED7 protein expression was associated with improved BC-specific survival within the whole cohort and ER+/luminal subgroup. Pooled MED7 gene expression data in the external validation cohort confirmed association with better survival, corroborating with the protein expression. On multivariate analysis, MED7 protein was independently predictive of longer BC-specific survival in the whole cohort and Luminal A subtype (p < 0.001).ConclusionsMED7 is an important prognostic marker in BC, particularly in ER+luminal subtypes, associated with improved survival and warrants future functional analysis.

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.

Castrate‐resistant prostate cancer (CRPC) is poorly characterized and heterogeneous and while the androgen receptor (AR) is of singular importance, other factors such as c‐Myc and the E2F family also play a role in later stage disease. HES6 is a transcription co‐factor associated with stem cell characteristics in neural tissue. Here we show that HES6 is up‐regulated in aggressive human prostate cancer and drives castration‐resistant tumour growth in the absence of ligand binding by enhancing the transcriptional activity of the AR, which is preferentially directed to a regulatory network enriched for transcription factors such as E2F1. In the clinical setting, we have uncovered a HES6‐associated signature that predicts poor outcome in prostate cancer, which can be pharmacologically targeted by inhibition of PLK1 with restoration of sensitivity to castration. We have therefore shown for the first time the critical role of HES6 in the development of CRPC and identified its potential in patient‐specific therapeutic strategies. Synopsis HES6 promotes castration resistance, maintains AR chromatin binding at a subset of sites in the absence of hormone stimulation and predicts poor outcome after prostatectomy. Inhibition of HES6‐responsive gene PLK1 enhances anti‐androgen sensitivity. HES6 promotes castration resistance in prostate cancer cells. HES6 maintains AR chromatin binding at a subset of sites in the absence of hormone stimulation. HES6‐associated genes predict poor clinical outcome after radical prostatectomy. HES6‐responsive gene PLK1 is highly expressed in a new hormone relapse TMA. Inhibition of PLK1 enhances sensitivity to anti‐androgens. Graphical Abstract HES6 promotes castration resistance, maintains AR chromatin binding at a subset of sites in the absence of hormone stimulation and predicts poor outcome after prostatectomy. Inhibition of HES6‐responsive gene PLK1 enhances anti‐androgen sensitivity.

Dissemination of primary tumor cells depends on migratory and invasive attributes. Here, we identify Navigator‐3 ( NAV3 ), a gene frequently mutated or deleted in human tumors, as a regulator of epithelial migration and invasion. Following induction by growth factors, NAV3 localizes to the plus ends of microtubules and enhances their polarized growth. Accordingly, NAV3 depletion trimmed microtubule growth, prolonged growth factor signaling, prevented apoptosis and enhanced random cell migration. Mathematical modeling suggested that NAV3‐depleted cells acquire an advantage in terms of the way they explore their environment. In animal models, silencing NAV3 increased metastasis, whereas ectopic expression of the wild‐type form, unlike expression of two, relatively unstable oncogenic mutants from human tumors, inhibited metastasis. Congruently, analyses of > 2,500 breast and lung cancer patients associated low NAV3 with shorter survival. We propose that NAV3 inhibits breast cancer progression by regulating microtubule dynamics, biasing directionally persistent rather than random migration, and inhibiting locomotion of initiated cells. Synopsis A new potential suppressor of metastasis, Navigator‐3, acts by modulating migration patterns and conferring a lower probability to locate tissue escape routes. Navigator‐3 (NAV3), a nerve‐navigating gene of worms and a gene frequently mutated or deleted in human tumors, was identified herein as a potential suppressor of metastasis, which associates with good prognosis of breast cancer patients. The NAV3 protein localizes to the growing end of microtubules, enhances their growth and augments the ability of cells to adhere to a migration course, while also slowing down migration rates. Animal studies indicate that these cellular actions of NAV3 overall reduce the capacity of tumor cells to form metastases, and mathematical modeling attributes this to lower probability to locate rare or transient targets, such as escape routes within tissues. NAV3 cancer mutations affect different protein domains; analysis of two point mutants uncovered a destabilization effect, suggesting loss of function in cancer. Graphical Abstract A new potential suppressor of metastasis, Navigator‐3, acts by modulating migration patterns and conferring a lower probability to locate tissue escape routes.

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. 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). 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). 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. Nottingham Hospitals Charity and the John and Lucille van Geest Foundation.