Subtype-specific micro-RNA expression signatures in breast cancer progression.

Robust markers of invasiveness may help reduce the overtreatment of in situ carcinomas. Breast cancer is a heterogeneous disease and biological mechanisms for carcinogenesis vary between subtypes. Stratification by subtype is therefore necessary to identify relevant and robust signatures of invasive disease. We have identified microRNA (miRNA) alterations during breast cancer progression in two separate datasets and used stratification and external validation to strengthen the findings. We analyzed two separate datasets (METABRIC and AHUS) consisting of a total of 186 normal breast tissue samples, 18 ductal carcinoma in situ (DCIS) and 1,338 invasive breast carcinomas. Validation in a separate dataset and stratification by molecular subtypes based on immunohistochemistry, PAM50 and integrated cluster classifications were performed. We propose subtype-specific miRNA signatures of invasive carcinoma and a validated signature of DCIS. miRNAs included in the invasive signatures include downregulation of miR-139-5p in aggressive subtypes and upregulation of miR-29c-5p expression in the luminal subtypes. No miRNAs were differentially expressed in the transition from DCIS to invasive carcinomas on the whole, indicating the need for subtype stratification. A total of 27 miRNAs were included in our proposed DCIS signature. Significant alterations of expression included upregulation of miR-21-5p and the miR-200 family and downregulation of let-7 family members in DCIS samples. The signatures proposed here can form the basis for studies exploring DCIS samples with increased invasive potential and serum biomarkers for in situ and invasive breast cancer.This work was performed as part of the EurocanPlatform which has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement No. 260791. Portions of this research (Venn diagram creator) were supported by the W.R. Wiley Environmental Molecular Science Laboratory, a national scientific user facility sponsored by the U.S. Department of Energy's Office of Biological and Environmental Research and located at PNNL. PNNL is operated by Battelle Memorial Institute for the U.S. Department of Energy under contract DE-AC05-76RL0 1830.This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1002/ijc.3014

Serum estradiol levels associated with specific gene expression patterns in normal breast tissue and in breast carcinomas

Abstract Background High serum levels of estradiol are associated with increased risk of postmenopausal breast cancer. Little is known about the gene expression in normal breast tissue in relation to levels of circulating serum estradiol. Methods We compared whole genome expression data of breast tissue samples with serum hormone levels using data from 79 healthy women and 64 breast cancer patients. Significance analysis of microarrays (SAM) was used to identify differentially expressed genes and multivariate linear regression was used to identify independent associations. Results Six genes (SCGB3A1, RSPO1, TLN2, SLITRK4, DCLK1, PTGS1) were found differentially expressed according to serum estradiol levels (FDR = 0). Three of these independently predicted estradiol levels in a multivariate model, as SCGB3A1 (HIN1) and TLN2 were up-regulated and PTGS1 (COX1) was down-regulated in breast samples from women with high serum estradiol. Serum estradiol, but none of the differentially expressed genes were significantly associated with mammographic density, another strong breast cancer risk factor. In breast carcinomas, expression of GREB1 and AREG was associated with serum estradiol in all cancers and in the subgroup of estrogen receptor positive cases. Conclusions We have identified genes associated with serum estradiol levels in normal breast tissue and in breast carcinomas. SCGB3A1 is a suggested tumor suppressor gene that inhibits cell growth and invasion and is methylated and down-regulated in many epithelial cancers. Our findings indicate this gene as an important inhibitor of breast cell proliferation in healthy women with high estradiol levels. In the breast, this gene is expressed in luminal cells only and is methylated in non-BRCA-related breast cancers. The possibility of a carcinogenic contribution of silencing of this gene for luminal, but not basal-like cancers should be further explored. PTGS1 induces prostaglandin E2 (PGE2) production which in turn stimulates aromatase expression and hence increases the local production of estradiol. This is the first report studying such associations in normal breast tissue in humans

Expression of BMI-1 and Mel-18 in breast tissue - a diagnostic marker in patients with breast cancer

<p>Abstract</p> <p>Background</p> <p>Polycomb Group (PcG) proteins are epigenetic silencers involved in maintaining cellular identity, and their deregulation can result in cancer. Expression of Mel-18 and Bmi-1 has been studied in tumor tissue, but not in adjacent non-cancerous breast epithelium. Our study compares the expression of the two genes in normal breast epithelium of cancer patients and relates it to the level of expression in the corresponding tumors as well as in breast epithelium of healthy women.</p> <p>Methods</p> <p>A total of 79 tumors, of which 71 malignant tumors of the breast, 6 fibroadenomas, and 2 DCIS were studied and compared to the reduction mammoplastic specimens of 11 healthy women. In addition there was available adjacent cancer free tissue for 23 of the malignant tumors. The tissue samples were stored in RNAlater, RNA was isolated to create expression microarray profile. These two genes were then studied more closely first on mRNA transcription level by microarrays (Agilent 44 K) and quantitative RT-PCR (TaqMan) and then on protein expression level using immunohistochemistry.</p> <p>Results</p> <p>Bmi-1 mRNA is significantly up-regulated in adjacent normal breast tissue in breast cancer patients compared to normal breast tissue from noncancerous patients. Conversely, mRNA transcription level of Mel-18 is lower in normal breast from patients operated for breast cancer compared to breast tissue from mammoplasty. When protein expression of these two genes was evaluated, we observed that most of the epithelial cells were positive for Bmi-1 in both groups of tissue samples, although the expression intensity was stronger in normal tissue from cancer patients compared to mammoplasty tissue samples. Protein expression of Mel-18 showed inversely stronger intensity in tissue samples from mammoplasty compared to normal breast tissue from patients operated for breast cancer.</p> <p>Conclusion</p> <p>Bmi-1 mRNA level is consistently increased and Mel-18 mRNA level is consistently decreased in adjacent normal breast tissue of cancer patients as compared to normal breast tissue in women having had reduction mammoplasties. Bmi-1/Mel-18 ratio can be potentially used as a tool for stratifying women at risk of developing malignancy.</p

Expression levels of uridine 5'-diphospho-glucuronosyltransferase genes in breast tissue from healthy women are associated with mammographic density

Introduction Mammographic density (MD), as assessed from film screen mammograms, is determined by the relative content of adipose, connective and epithelial tissue in the female breast. In epidemiological studies, a high percentage of MD confers a four to six fold risk elevation of developing breast cancer, even after adjustment for other known breast cancer risk factors. However, the biologic correlates of density are little known. Methods Gene expression analysis using whole genome arrays was performed on breast biopsies from 143 women; 79 women with no malignancy (healthy women) and 64 newly diagnosed breast cancer patients, both included from mammographic centres. Percent MD was determined using a previously validated, computerized method on scanned mammograms. Significance analysis of microarrays (SAM) was performed to identify genes influencing MD and a linear regression model was used to assess the independent contribution from different variables to MD. Results SAM-analysis identified 24 genes differentially expressed between samples from breasts with high and low MD. These genes included three uridine 5'-diphospho-glucuronosyltransferase (UGT) genes and the oestrogen receptor gene (ESR1). These genes were down-regulated in samples with high MD compared to those with low MD. The UGT gene products, which are known to inactivate oestrogen metabolites, were also down-regulated in tumour samples compared to samples from healthy individuals. Several single nucleotide polymorphisms (SNPs) in the UGT genes associated with the expression of UGT and other genes in their vicinity were identified. Conclusions Three UGT enzymes were lower expressed both in breast tissue biopsies from healthy women with high MD and in biopsies from newly diagnosed breast cancers. The association was strongest amongst young women and women using hormonal therapy. UGT2B10 predicts MD independently of age, hormone therapy and parity. Our results indicate that down-regulation of UGT genes in women exposed to female sex hormones is associated with high MD and might increase the risk of breast cancer

Gene expression profiles of breast biopsies from healthy women identify a group with claudin-low features

Background Increased understanding of the variability in normal breast biology will enable us to identify mechanisms of breast cancer initiation and the origin of different subtypes, and to better predict breast cancer risk. Methods Gene expression patterns in breast biopsies from 79 healthy women referred to breast diagnostic centers in Norway were explored by unsupervised hierarchical clustering and supervised analyses, such as gene set enrichment analysis and gene ontology analysis and comparison with previously published genelists and independent datasets. Results Unsupervised hierarchical clustering identified two separate clusters of normal breast tissue based on gene-expression profiling, regardless of clustering algorithm and gene filtering used. Comparison of the expression profile of the two clusters with several published gene lists describing breast cells revealed that the samples in cluster 1 share characteristics with stromal cells and stem cells, and to a certain degree with mesenchymal cells and myoepithelial cells. The samples in cluster 1 also share many features with the newly identified claudin-low breast cancer intrinsic subtype, which also shows characteristics of stromal and stem cells. More women belonging to cluster 1 have a family history of breast cancer and there is a slight overrepresentation of nulliparous women in cluster 1. Similar findings were seen in a separate dataset consisting of histologically normal tissue from both breasts harboring breast cancer and from mammoplasty reductions. Conclusion This is the first study to explore the variability of gene expression patterns in whole biopsies from normal breasts and identified distinct subtypes of normal breast tissue. Further studies are needed to determine the specific cell contribution to the variation in the biology of normal breasts, how the clusters identified relate to breast cancer risk and their possible link to the origin of the different molecular subtypes of breast cancer

Comparable cancer-relevant mutation profiles in synchronous ductal carcinoma in situ and invasive breast cancer

Background: Ductal carcinoma in situ (DCIS) comprises a diverse group of preinvasive lesions in the breast and poses a considerable clinical challenge due to lack of markers of progression. Genomic alterations are to a large extent similar in DCIS and invasive carcinomas, although differences in copy number aberrations, gene expression patterns, and mutations exist. In mixed tumors with synchronous invasive breast cancer (IBC) and DCIS, it is still unclear to what extent invasive tumor cells are directly derived from the DCIS cells. Aim: Our aim was to compare cancer-relevant mutation profiles of different cellular compartments in mixed DCIS/IBC and pure DCIS tumors. Methods and results: We performed targeted sequencing of 50 oncogenes in microdissected tissue from three different epithelial cell compartments (in situ, invasive, and normal adjacent epithelium) from 26 mixed breast carcinomas. In total, 44 tissue samples (19 invasive, 16 in situ, 9 normal) were subjected to sequencing using the Ion Torrent platform and the AmpliSeq Cancer Hotspot Panel v2. For comparison, 10 additional, pure DCIS lesions were sequenced. Across all mixed samples, we detected 23 variants previously described in cancer. The most commonly affected genes were TP53, PIK3CA, and ERBB2. The PIK3CA:p.H1047R variant was found in nine samples from six patients. Most variants detected in invasive compartments were also found in the corresponding in situ cell compartment indicating a clonal relationship between the tumor stages. A lower frequency of variants were observed in pure DCIS lesions. Conclusion: Similar mutation profiles between in situ and invasive cell compartments indicate a similar origin of the two tumor stages in mixed breast tumors. The lower number of potential driver variants found in pure DCIS compared with the in situ cell compartments of mixed tumors may imply that pure DCIS is captured earlier in the path of progression to invasive disease

Proline Conformation-Dependent Antimicrobial Activity of a Proline-Rich Histone H1 N-Terminal Peptide Fragment Isolated from the Skin Mucus of Atlantic Salmon

A 30-residue N-terminally acetylated peptide derived from the N-terminal part of histone H1 was identified as the dominant antimicrobial peptide in skin mucus from Atlantic salmon (Salmo salar). The peptide (termed salmon antimicrobial peptide [SAMP H1]) was purified to homogeneity by a combination of reversed-phase and cation-exchange chromatographies. By Edman degradation of the deacetylated peptide and by sequencing of the PCR-amplified DNA that encodes the peptide, the complete amino acid sequence was determined to be AEVAPAPAAAAPAKAPKKKAAAKPKKAGPS. The theoretical molecular weight of N-terminally acetylated SAMP H1 was calculated to be 2,836, which is the same as that determined by matrix-assisted laser desorption ionization mass spectrometry. The peptide was active against both gram-negative and -positive bacteria. The N-terminal acetyl group was not necessary for activity since deacetylation did not reduce the activity. A synthetic peptide whose sequence was identical to that of the isolated fragment was initially inactive but could be activated by binding it to a cation-exchange column. Treatment of the synthetic peptide when it was bound to the exchange column with peptidylproline cis-trans-isomerase increased the amount of active peptide, indicating that isomerization of the proline peptide bond(s) was necessary for activation of the synthetic peptide. Comparison of the active and inactive forms by circular dichroism and chromatographic analyses suggests that the active form, both the natural and the synthetic forms, is more structured, condensed, and rigid than the inactive form, which has a more nonstructured conformation. This work shows for the first time the importance of proline isomers in the activity of an antimicrobial peptide

Strong Synergy between a Eukaryotic Antimicrobial Peptide and Bacteriocins from Lactic Acid Bacteria

The antimicrobial effect obtained upon combining the prokaryotic antimicrobial peptides (AMPs; more commonly referred to as bacteriocins) pediocin PA-1, sakacin P, and curvacin A (all produced by lactic acid bacteria [LAB]) with the eukaryotic AMP pleurocidin (from fish) has been investigated. The three LAB AMPs alone were active against gram-positive Listeria ivanovii bacteria at nanomolar concentrations, whereas they were inactive against gram-negative Escherichia coli bacteria. Pleurocidin alone was active against both of these types of bacteria at micromolar concentrations. Little if any synergy between the LAB AMPs and pleurocidin against the gram-positive L. ivanovii strain was obtained. In contrast, the LAB AMPs and pleurocidin acted highly synergistically against the gram-negative E. coli strain. Nanomolar concentrations of LAB AMPs increased the growth inhibitory potency of pleurocidin by about fourfold. When micromolar concentrations of LAB AMPs were combined with 2 μg of pleurocidin/ml, 100% growth inhibition was attained, whereas pleurocidin alone at a concentration of 2 μg/ml gave no growth inhibition. Most noteworthy, when high concentrations (128 μg/ml) of pleurocidin in the absence of LAB AMPs were used over a long period of incubation (1 week), some growth of E. coli was observed, whereas 16 μg of pleurocidin/ml completely abolished growth in the presence of 64 to 128 ng of LAB AMPs/ml over the same period of time. The results clearly demonstrate that combining eukaryotic and prokaryotic AMPs can greatly increase the specific activity and broaden the target-cell range of these peptides