Purpose Genomic profiling studies suggest triple-negative breast cancer (TNBC) is a heterogeneous disease. In this study we sought to define TNBC subtypes and identify subtype-specific markers and targets. Patients and Methods RNA and DNA profiling analyses were conducted on 198 TNBC tumors (ER-negativity defined as Allred Scale value ≤2) with >50% cellularity (discovery set: n=84; validation set: n=114) collected at Baylor College of Medicine. An external data set of 7 publically-accessible TNBC studies was used to confirm results. DNA copy number, disease-free survival (DFS) and disease-specific survival (DSS) were analyzed independently using these datasets. Results We identified and confirmed four distinct TNBC subtypes: (1) Luminal-AR (LAR); 2) Mesenchymal (MES); 3) Basal-Like Immune-Suppressed (BLIS), and 4) Basal-Like Immune-Activated (BLIA). Of these, prognosis is worst for BLIS tumors and best for BLIA tumors for both DFS (logrank test p=0.042 and 0.041, respectively) and DSS (logrank test p=0.039 and 0.029, respectively). DNA copy number analysis produced two major groups (LAR and MES/BLIS/BLIA), and suggested gene amplification drives gene expression in some cases (FGFR2 (BLIS)). Putative subtype-specific targets were identified: 1) LAR: androgen receptor and the cell surface mucin MUC1; 2) MES: growth factor receptors (PDGF receptor A; c-Kit); 3) BLIS: an immune suppressing molecule (VTCN1); and 4) BLIA: Stat signal transduction molecules and cytokines. Conclusion There are four stable TNBC subtypes characterized by the expression of distinct molecular profiles that have distinct prognoses. These studies identify novel subtype-specific targets that can be targeted in the future for effective treatment of TNBCs.

Triple-negative breast cancers (TNBCs) are aggressive with no effective targeted therapies. A combined database analysis identified 32 inflammation-related genes differentially expressed in TNBCs, 10 proved critical for anchorage-independent growth. In TNBC cells a LPA-LPAR2-EZH2 NF-kappaB signaling cascade was essential for expression of IL-6, IL-8 and CXCL1. Concurrent inhibition of IL-6 and IL-8 expression dramatically inhibited colony formation and cell survival in vitro and stanched tumor engraftment and growth in vivo. A Cox multivariable analysis of patient specimens revealed that IL-6 and IL-8 expression predicted patient survival times. Together these findings offer a rationale for dual inhibition of IL-6/IL-8 signaling as a therapeutic strategy to improve outcomes for TNBC patients.

Background: Fetal programming describes the theory linking environmental conditions during embryonic and fetal development with risk of diseases later in life. Environmental insults in utero may lead to changes in epigenetic mechanisms potentially affecting fetal development.Objectives: We examined associations between in utero exposures, infant growth, and methylation of repetitive elements and gene-associated DNA in human term placenta tissue samples.Methods: Placental tissues and associated demographic and clinical data were obtained from subjects delivering at Women and Infants Hospital in Providence, Rhode Island (USA). Methylation levels of long interspersed nuclear element-1 (LINE-1) and the Alu element AluYb8 were determined in 380 placental samples from term deliveries using bisulfite pyrosequencing. Genomewide DNA methylation profiles were obtained in a subset of 184 samples using the Illumina Infinium HumanMethylation27 BeadArray. Multiple linear regression, model-based clustering methods, and gene set enrichment analysis examined the association between birth weight percentile, demographic variables, and repetitive element methylation and gene-associated CpG locus methylation.Results: LINE-1 and AluYb8 methylation levels were found to be significantly positively associated with birth weight percentile (p = 0.01 and p < 0.0001, respectively) and were found to differ significantly among infants exposed to tobacco smoke and alcohol. Increased placental AluYb8 methylation was positively associated with average methylation among CpG loci found in polycomb group target genes; developmentally related transcription factor binding sites were overrepresented for differentially methylated loci associated with both elements.Conclusions: Our results suggest that repetitive element methylation markers, most notably AluYb8 methylation, may be susceptible to epigenetic alterations resulting from the intrauterine environment and play a critical role in mediating placenta function, and may ultimately inform on the developmental basis of health and disease.

Purpose The human epigenome is profoundly altered in cancers, with a characteristic loss of methylation in repetitive regions and concomitant accumulation of gene-promoter methylation. The degree to which these processes are coordinated is unclear so we investigated both in head and neck squamous cell carcinomas. Experimental Design Global methylation was measured using the luminometric methylation assay (LUMA), and pyrosequencing of LINE-1Hs and AluYb8 repetitive elements in a series of 138 tumors. We also measured methylation of over 27,000 CpG loci with the Illumina HumanMethylation27 microarray (n=91). Results LINE-1 methylation was significantly associated with LUMA and Infinium loci methylation (Spearman’s rho=0.52/rho=0.56, both p<0.001), but not that of AluYb8. Methylation of LINE-1, AluYb8, and Infinium loci differed by tumor site (each Kruskal-Wallis p<0.05). Also, LINE-1 and LUMA methylation were associated with HPV16 E6 serology (each Mann-Whitney p<0.05). Comparing LINE-1 methylation to gene-associated methylation, we identified a distinct subset of CpG loci with significant hypermethylation associated with LINE-1 hypomethylation. An investigation of sequence features for these CpG loci revealed that they were significantly less likely to reside in repetitive elements (GSEA p<0.02), enriched in CpG islands (p<0.001), and were proximal to transcription factor binding sites (p<0.05). We validated the top CpG loci that had significant hypermethylation associated with LINE-1 hypomethylation (at EVI2A, IFRD1, KLHL6, and PTPRCAP) by pyrosequencing independent tumors. Conclusions These data indicate that global hypomethylation and gene-specific methylation processes are associated in a sequence-dependent manner, and that clinical characteristics and exposures leading to HNSCC may be influencing these processes.