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Supplementary Material: Figure S1: Nuclear localisation and fluorescent properties of FRET constructs Figure S2: Quantification of FRET experiment Figure S3: Relative mRNA expression of ESR1 target genes Figure S4: Characterisation of MCF-7 clones overexpressing NFIB and YBX1 Figure S5: Separated growth curves shown in Figure 4 Figure S6: Kaplan-Meier survival curves for breast cancer cases stratified by YBX1 expression Figure S7: Response of breast cancer PDX models to tamoxifen treatment Table S1: Primers used in RT-PCR Table S2: Antibodies used in Western blots

Full data set obtained in RIME experiment.

Two opposing clusters of transcription factors (TF) have been associated with the differential risks of estrogen receptor positive or negative breast cancers, but the mechanisms underlying the opposing functions of the two clusters are undefined. In this study, we identified NFIB and YBX1 as novel interactors of the estrogen receptor (ESR1). NFIB and YBX1 are both risk TF associated with progression of ESR1-negative disease. Notably, they both interacted with the ESR1-FOXA1 complex and inhibited the transactivational potential of ESR1. Moreover, signaling through FGFR2, a known risk factor in breast cancer development, augmented these interactions and further repressed ESR1 target gene expression. We therefore show that members of two opposing clusters of risk TFs associated with ESR1-positive and -negative breast cancer can physically interact. We postulate that this interaction forms a toggle between two developmental pathways affected by FGFR2 signaling, possibly offering a junction to exploit therapeutically.Significance: Binding of the transcription factors NFIB and YBX1 to the estrogen receptor can promote an estrogen-independent phenotype that can be reverted by inhibiting FGFR2 signaling. Cancer Res; 78(2); 410–21. ©2017 AACR.

BackgroundBreast cancer (BC) genome-wide association studies (GWAS) have identified hundreds of risk-loci that require novel approaches to reveal the causal variants and target genes within them. As causal variants are most likely regulators of gene expression, we hypothesize that their identification is facilitated by pinpointing the variants with greater regulatory potential within risk-loci.MethodsWe performed genome-wide differential allelic expression (DAE) analysis using microarrays data from 64 normal breast tissue samples. Then, we mapped the variants associated with DAE (daeQTLs) and intersected these with GWAS data to reveal candidate risk regulatory variants. Finally, we validated our approach by functionally analysing the 5q14.1 breast cancer risk-locus.ResultsWe found widespread gene expression regulation by cis-acting variants in breast tissue, with 80% of coding and non-coding expressed genes displaying DAE (daeGenes). We identified over 23K daeQTLs for 2753 (16%) daeGenes, including at 154 known BC risk- loci. And in 31 of these risk-loci, we found risk-associated variant(s) and daeQTLs in strong linkage disequilibrium suggesting that the risk-causing variants are cis-regulatory, and in 27 risk-loci we propose 37 candidate target genes. As validation, we identified five candidate causal variants at the 5q14.1 risk-locus targeting the ATG10, RPS23, and ATP6AP1L genes, likely via modulation of miRNA binding, alternative transcription, and transcription factor binding.ConclusionOur study shows the power of DAE analysis and daeQTL mapping to identify causal regulatory variants and target genes at BC risk loci, including those with complex regulatory landscapes, and provides a genome-wide resource of variants associated with DAE for future functional studies.

Lung cancer is the leading cause of cancer-related death in the world. In contrast to many other cancers, a direct connection to modifiable lifestyle risk in the form of tobacco smoke has long been established. More than 50% of all smoking-related lung cancers occur in former smokers, often many years after smoking cessation. Despite extensive research, the molecular processes for persistent lung cancer risk are unclear.To examine whether risk stratification in the clinic and in the general population can be improved upon by the addition of genetic data, and to explore the mechanisms of the persisting risk in former smokers, we have analysed transcriptomic data from accessible airway tissues of 487 subjects, including healthy volunteers and clinic patients of different smoking status. We developed a model to assess smoking associated gene expression changes and their reversibility after smoking is stopped, comparing healthy subjects to clinic patients with and without lung cancer. We find persistent smoking-associated immune alterations to be a hallmark of the clinic patients. Integrating previous GWAS data using a transcriptional network approach, we demonstrate that the same immune and interferon related pathways are strongly enriched for genes linked to known genetic risk factors, demonstrating a causal relationship between immune alteration and lung cancer risk. Finally, we used accessible airway transcriptomic data to derive a non-invasive lung cancer risk classifier.Our results provide initial evidence for germline-mediated personalised smoke injury response and risk in the general population, with potential implications for managing long-term lung cancer incidence and mortality.

Motivation Dendrogram is a classical diagram for visualizing binary trees. Although efficient to represent hierarchical relations, it provides limited space for displaying information on the leaf elements, especially for large trees. Results Here, we present TreeAndLeaf, an R/Bioconductor package that implements a hybrid layout strategy to represent tree diagrams with focus on the leaves. The TreeAndLeaf package combines force-directed graph and tree layout algorithms using a single visualization system, allowing projection of multiple layers of information onto a graph-tree diagram. The Supplementary Information provides two case studies that use breast cancer data from epidemiological and experimental studies. Availability and implementation TreeAndLeaf is written in the R language, and is available from the Bioconductor project at http://bioconductor.org/packages/TreeAndLeaf/ (version≥1.4.2). Supplementary information Supplementary data are available at Bioinformatics online.

A breast-ovarian cancer susceptibility gene, BRCA1, which is responsible for disease in approximately 45% of breast cancer families and most families that contain breast and ovarian cancer, has been assigned by genetic linkage to 17q12-21. Here, we report the analysis of three marker-disease recombinants in families that contain breast and ovarian cancer, two of which strongly suggest a location for BRCA1 telomeric to D17S702, a microsatellite polymorphism, and a third which suggests a location centromeric to EDH17B, the gene encoding estradiol-17B dehydrogenase. If the interpretation of these recombinants is correct, the results localise BRCA1 to an interval of < or = 1 cM.

Recent studies based on genome-wide association, linkage, and admixture scan analysis have reported associations of various genetic variants in 8q24 with susceptibility to breast, prostate, and colorectal cancer. This locus lies within a 1.18-Mb region that contains no known genes but is bounded at its centromeric end by FAM84B and at its telomeric end by c-MYC, two candidate cancer susceptibility genes. To investigate the associations of specific loci within 8q24 with specific cancers, we genotyped the nine previously reported cancer-associated single-nucleotide polymorphisms across the region in four case-control sets of prostate (1854 case subjects and 1894 control subjects), breast (2270 case subjects and 2280 control subjects), colorectal (2299 case subjects and 2284 control subjects), and ovarian (1975 case subjects and 3411 control subjects) cancer. Five different haplotype blocks within this gene desert were specifically associated with risks of different cancers. One block was solely associated with risk of breast cancer, three others were associated solely with the risk of prostate cancer, and a fifth was associated with the risk of prostate, colorectal, and ovarian cancer, but not breast cancer. We conclude that there are at least five separate functional variants in this region.

Misdiagnosis of a germline mutation associated with an inherited disease syndrome can have serious implications for the clinical management of patients. A false negative diagnosis (mutation missed by genetic screening) limits decision making about intervention strategies within families. More serious is the consequence of a false positive diagnosis (genetic test suggesting a mutation is present when it is not). This could lead to an individual, falsely diagnosed as a mutation carrier, undergoing unnecessary clinical intervention, possibly involving risk-reducing surgery. As part of screening 283 ovarian cancer families for BRCA1 mutations, we used two different methods (mutation specific PCR and multiplex ligation-dependent probe amplification) to screen for a known rearrangement mutation L78833.1:g.44369_50449dup (ins6kbEx13). We found false positive and false negative results in several families. We then tested 61 known carriers or non-carriers from an epidemiological study of BRCA1 and BRCA2 mutation carriers (the EMBRACE study). These data highlight the need for caution when interpreting analyses of the ins6kbEx13 mutation and similar mutations, where characterising the exact sequence alteration for a deleterious mutation is not a part of the routine genetic test.

STK15 is a putative oncogene that codes for a centrosome-associated, serine/threonine kinase, the normal function of which is to ensure accurate segregation of chromosomes during mitosis. Amplification of STK15 has been reported in ovarian tumors, suggesting a role in ovarian cancer pathology. STK15 is polymorphic with two single nucleotide substitutions (449t/a and 527g/a) in evolutionarily conserved regions causing amino acid changes (F31I and V57I). Two other nucleotide substitutions (287c/g and 1891g/c) of unknown significance are in 5' and 3' untranslated regions (UTR), respectively. To learn more about the involvement of STK15 in ovarian cancer, we genotyped and haplotyped these polymorphisms in three population-based ovarian cancer case-control studies from the United Kingdom, United States, and Denmark with 1,821 combined cases and 2,467 combined controls and calculated risks for developing ovarian cancer. Genotypes of individual polymorphisms in control groups of the United Kingdom, United States, and Denmark conformed to Hardy-Weinberg equilibrium. In combined cases and combined controls, rare allele frequencies were 0.23 and 0.21 for I31, 0.16 and 0.17 for I57, 0.08 and 0.07 for 5' UTR g, and 0.25 and 0.24 for 3' UTR c, respectively. Using FF common homozygotes of F31I as comparator, there was increased ovarian cancer risk to FI heterozygotes (odds ratio, 1.18; 95% confidence interval, 1.01-1.36), II homozygotes (odds ratio, 1.25; 95% confidence interval, 0.89-1.75), and I31 allele carriers (odds ratio, 1.17; 95% confidence interval, 1.02-1.35) in the combined group data. For either V57I, 5' UTR C/G, or 3' UTR G/C, all genotypic ovarian cancer risks were essentially in unity relative to their respective common homozygotes, VV, cc, or gg. Haplotype analysis of combined group data revealed seven haplotypes with frequencies between 0.02 and 0.5, with c-F-V-g the most common. None of the haplotype-specific risks significantly differed from unity relative to c-F-V-g. These results suggest a model of dominant inheritance of ovarian cancer risk by the I31 allele of F31I and that the I31 allele may be a common ovarian cancer susceptibility allele of low penetrance.

We described previously a thyroid phenotype for transgenic mice expressing an activated Ret oncogene driven from a human calcitonin promoter. Medullary thyroid carcinomas (MTC), a tumor of the thyroid parafollicular C cells, occur in this transgenic line with a pathology analogous to that seen in patients with multiple endocrine neoplasia type 2 (MEN2). When the transgene was introgressed onto four different genetic backgrounds, between 0 and 98% of transgenic progeny developed thyroid tumors by 10 months of age, indicating that tumor penetrance could be modulated by genetic background. Furthermore, tumors on the CBA/ca and C57BL/6J backgrounds were significantly larger than those arising in BALB/c transgenic mice. These results are relevant to human MEN2 disease, because this model system may be used to study genes modifying thyroid tumor penetrance in the dominantly inherited human cancer syndrome.

There is evidence that transforming growth factor (TGF)beta acts as a suppressor of tumor initiation but also as a promoter of tumor progression when the antiproliferative effect of the TGFbeta signaling pathway has been overridden by other oncogenic mutations. Several somatic mutations that disrupt the TGFbeta-SMAD signaling pathway have been reported in human breast tumors. We have examined the association between single nucleotide polymorphisms (SNPs) in the TGFbeta1 gene and the incidence of invasive breast cancer in three case-control series, with a maximum of 3987 patients and 3867 controls, median age approximately 50 years, and range 22-92 years. The promoter SNP, C-509T, and the T +29C signal-peptide SNP (encoding Leu10Pro) are in strong linkage disequilibrium. They are both significantly associated with increased incidence of invasive breast cancer in a recessive manner [odds ratios: (TT versus C-carrier), 1.25; 95% confidence intervals 1.06-1.48; P = 0.009 and (ProPro versus Leu-carrier), 1.21; 95% confidence intervals 1.05-1.37; P = 0.01]. The G-800A SNP was not significantly associated with incidence of breast cancer. The C-509T SNP is not contained within a known consensus sequence for a promoter regulatory element and therefore unlikely to affect TGFbeta1 expression, whereas the Leu10Pro signal peptide substitution potentially affects TGFbeta1 secretion. Transfections of HeLa cells with constructs encoding either the Pro or Leu forms of TGFbeta1 and driven by the cytomegalovirus promoter indicate that the signal peptide with Pro at residue 10 causes a 2.8-fold increase in secretion compared with the Leu form. These data indicate that the allele encoding Pro10 is associated with increased rates of TGFbeta1 secretion and with increased incidence of invasive breast cancer for the population samples described. It is estimated that 3% of all breast cancer cases may be attributable to Pro10 homozygosity.

Somatic genetic alterations in tumors are known to correlate with survival, but little is known about the prognostic significance of germ-line variation. We assessed the effect of germ-line variation on survival among women with breast cancer participating in a British population-based study. Up to 2430 cases for whom current vital status data were available were screened for BRCA1/2 mutations and genotyped for polymorphisms in 22 DNA repair, hormone metabolism, carcinogen metabolism, and other genes. The effect of genotype on outcome was assessed by Cox regression analysis. The largest effect was observed for the silent polymorphism D501D (tc) in LIG4, a gene involved in DNA double-strand break repair. The estimated hazard ratio (HR) in cc homozygotes relative to tt homozygotes was 4.0 (95% confidence interval, 2.1-7.7; P = 0.002), and this effect remained after stratification by stage, grade, and tumor type [HR, 4.2 (1.8-9.4); P = 0.01]. Total length of a CYP19 IVS4 (ttta)(n) repeat was also associated with survival [HR, 0.9 (0.8-1.0); P = 0.01], but this became nonsignificant after stratification by stage, grade, and tumor type. Poorer survival was observed for 10 BRCA1 mutation carriers [HR, 4.1 (1.3-13); P = 0.047]; however, after adjustment for known prognostic factors, the HR estimate decreased to 2.0 and became nonsignificant (P = 0.4). CYP17 (P = 0.05) and TP53 (P = 0.06) polymorphisms showed marginally significant associations in unstratified analyses. No effect on survival was seen for polymorphisms in ATM, BRCA1/2, CHK2, KU70, NBS1, RAD51, RAD52, XRCC3, AR, COMT, NQO1, VDR, ADH3, CYP1A1, GSTP1, TGF-beta, or CDH1. Even if confirmed, the prognostic markers identified in this study are unlikely to replace current markers of prognosis such as estrogen receptor status. However, our results demonstrate the potential of the analysis of germ-line variation to provide insight into the biological determinants of response to treatment and prognosis in breast cancer.

Cancer genetics has for many years focused on mutational events that have their primary effect within the cancer cell. Recently that focus has widened, with evidence of the importance of epigenetic events and of cellular interactions in cancer development. The role of common genetic variation in determining the range of individual susceptibility within the population is increasingly recognized, and will be addressed using information from the Human Genome Project. These new research directions will highlight determinants of cancer that lie outside the cancer cell, suggest new targets for intervention, and inform the design of strategies for prevention in groups at increased risk.

Multiple endocrine neoplasia type 2A (MEN 2A) is a dominantly inherited cancer syndrome that affects tissues derived from neural ectoderm. It is characterized by medullary thyroid carcinoma (MTC) and phaeochromocytoma. The MEN2A gene has recently been localized by a combination of genetic and physical mapping techniques to a 480-kilobase region in chromosome 10q11.2 (refs 2,3). The DNA segment encompasses the RET proto-oncogene, a receptor tyrosine kinase gene expressed in MTC and phaeochromocytoma and at lower levels in normal human thyroid. This suggested RET as a candidate for the MEN2A gene. We have identified missense mutations of the RET proto-oncogene in 20 of 23 apparently distinct MEN 2A families, but not in 23 normal controls. Further, 19 of these 20 mutations affect the same conserved cysteine residue at the boundary of the RET extracellular and transmembrane domains.

Clonal analysis of whole-mount preparations of entire retinal pigment epithelium (RPE), using SWR↔C57BL/6JLac and DDK↔C3H/Bi mouse aggregation chimaeras in which one of the two parental components predominated, revealed a markedly non-random spatial arrangement of patch (clone) sizes. Single-cell and small patches predominated in an area around the optic nerve head while large patches occurred most frequently near the periphery. Mechanisms are discussed which may explain these results. Patch size frequency distributions were concave and skewed. Singletons were the most frequent size class, but a wide range of sizes and a smaller number of much larger patches were also always found. The results preclude the use of statistical methods previously employed to calculate clone sizes from the geometric means of observed patch sizes. Instead, the median and interquartile range may provide the best summary of the observed patch size frequency distributions. Our findings support a stochastic model of tissue growth.

We used staining of tissue sections by lectin conjugates to screen inbred strains of mice for polymorphisms which could be used as histological markers of chimaerism. We found one polymorphism, which involves reciprocal patterns of expression of binding sites for the N-acetyl-galactosamine-binding lectins from Dolichos biflorus (DBA), Helixpomatia (HPA) and Wisteria floribunda (WFA) on intestinal epithelium and vascular endothelium. The polymorphism is due to alleles at a single locus, designated Dlb-1 (for Dolichos lectin binding). Of 29 inbred strains examined, 3 are D1b-1a (type strain RIII-ro; gut epithelium - ve, vascular endothelium + ve), and 26 are D1b-1b (type strain C57BL/6J; gut epithelium + ve, vascular endothelium − ve). In RIII-ro and C57BL/6J embryos, the polymorphic difference is not clearly present until day 11 of gestation. Before then, embryos of both strains express binding sites on gut epithelium and on endothelium. The temporal and tissue-specific patterns of expression of lectin-binding sites may result from differences in expression of an N-acetyl galactosaminosyl transferase. If so, elucidation of the genetic basis of the polymorphism might provide an insight into the mechanisms of developmental regulation of glycosyltransferase activity.

The epithelium of each individual intestinal crypt in adult mouse aggregation chimaeras is composed of cells of a single parental genotype (Ponder et al. 1985). Using a carbohydrate polymorphism recognized by Dolichos biflorus agglutinin as a strain-specific marker on entire sheets of intestinal mucosa, we have analysed the two-dimensional mosaic patterns of patches of the chimaeric intestinal crypt population. The relative proportions of each genotype varied greatly along the length of any one intestine. In chimaeras with highly unbalanced proportions, the minority component occurred as discrete patches. Patches of single or a few crypts were most frequent, but a smaller number of much larger patches was always present. The size frequency distribution of discrete patches was highly concave and departed significantly from a geometric distribution (a model for non-differential proliferation), but fitted the more skewed negative binomial model. The data are consistent with the interpretation that most progenitor crypts never or rarely divide, while a minority proliferate to a greater extent. We discuss ways in which our system could be analysed further to examine this interpretation. Our results also support Whitten’s (1978) conclusion from a computer simulation that the mean patch size, as it has previously been used in statistical analyses of chimaeric tissue, ‘is not a reliable statistic on which to judge mosaicism’.