Complex molecular profile of DNA repair genes in epithelial ovarian carcinoma patients with different sensitivity to platinum-based therapy.

Epithelial ovarian carcinoma (EOC) is known for high mortality due to diagnosis at advanced stages and frequent therapy resistance. Previous findings suggested that the DNA repair system is involved in the therapeutic response of cancer patients and DNA repair genes are promising targets for novel therapies. This study aimed to address complex inter-relations among gene expression levels, methylation profiles, and somatic mutations in DNA repair genes and EOC prognosis and therapy resistance status. We found significant associations of DUT expression with the presence of peritoneal metastases in EOC patients. The high-grade serous EOC subtype was enriched with TP53 mutations compared to other subtypes. Furthermore, somatic mutations in XPC and PRKDC were significantly associated with worse overall survival of EOC patients, and higher FAAP20 expression in platinum-resistant than platinum-sensitive patients was observed. We found higher methylation of RAD50 in platinum-resistant than in platinum-sensitive patients. Somatic mutations in BRCA1 and RAD9A were significantly associated with higher RBBP8 methylation in platinum-sensitive compared to platinum-resistant EOC patients. In conclusion, we discovered associations of several candidate genes from the DNA repair pathway with the prognosis and platinum resistance status of EOC patients, which deserve further validation as potential predictive biomarkers.

Subtype and cell type specific expression of lncRNAs provide insight into breast cancer.

Long non-coding RNAs (lncRNAs) are involved in breast cancer pathogenesis through chromatin remodeling, transcriptional and post-transcriptional gene regulation. We report robust associations between lncRNA expression and breast cancer clinicopathological features in two population-based cohorts: SCAN-B and TCGA. Using co-expression analysis of lncRNAs with protein coding genes, we discovered three distinct clusters of lncRNAs. In silico cell type deconvolution coupled with single-cell RNA-seq analyses revealed that these three clusters were driven by cell type specific expression of lncRNAs. In one cluster lncRNAs were expressed by cancer cells and were mostly associated with the estrogen signaling pathways. In the two other clusters, lncRNAs were expressed either by immune cells or fibroblasts of the tumor microenvironment. To further investigate the cis-regulatory regions driving lncRNA expression in breast cancer, we identified subtype-specific transcription factor (TF) occupancy at lncRNA promoters. We also integrated lncRNA expression with DNA methylation data to identify long-range regulatory regions for lncRNA which were validated using ChiA-Pet-Pol2 loops. lncRNAs play an important role in shaping the gene regulatory landscape in breast cancer. We provide a detailed subtype and cell type-specific expression of lncRNA, which improves the understanding of underlying transcriptional regulation in breast cancer.

Epigenetic alterations at distal enhancers are linked to proliferation in human breast cancer.

Aberrant DNA methylation is an early event in breast carcinogenesis and plays a critical role in regulating gene expression. Here, we perform genome-wide expression-methylation Quantitative Trait Loci (emQTL) analysis through the integration of DNA methylation and gene expression to identify disease-driving pathways under epigenetic control. By grouping the emQTLs using biclustering we identify associations representing important biological processes associated with breast cancer pathogenesis including regulation of proliferation and tumor-infiltrating fibroblasts. We report genome-wide loss of enhancer methylation at binding sites of proliferation-driving transcription factors including CEBP-ß, FOSL1, and FOSL2 with concomitant high expression of proliferation-related genes in aggressive breast tumors as we confirm with scRNA-seq. The identified emQTL-CpGs and genes were found connected through chromatin loops, indicating that proliferation in breast tumors is under epigenetic regulation by DNA methylation. Interestingly, the associations between enhancer methylation and proliferation-related gene expression were also observed within known subtypes of breast cancer, suggesting a common role of epigenetic regulation of proliferation. Taken together, we show that proliferation in breast cancer is linked to loss of methylation at specific enhancers and transcription factor binding and gene activation through chromatin looping.

Crosstalk between microRNA expression and DNA methylation drives the hormone-dependent phenotype of breast cancer.

BACKGROUND: Abnormal DNA methylation is observed as an early event in breast carcinogenesis. However, how such alterations arise is still poorly understood. microRNAs (miRNAs) regulate gene expression at the post-transcriptional level and play key roles in various biological processes. Here, we integrate miRNA expression and DNA methylation at CpGs to study how miRNAs may affect the breast cancer methylome and how DNA methylation may regulate miRNA expression. METHODS: miRNA expression and DNA methylation data from two breast cancer cohorts, Oslo2 (n = 297) and The Cancer Genome Atlas (n = 439), were integrated through a correlation approach that we term miRNA-methylation Quantitative Trait Loci (mimQTL) analysis. Hierarchical clustering was used to identify clusters of miRNAs and CpGs that were further characterized through analysis of mRNA/protein expression, clinicopathological features, in silico deconvolution, chromatin state and accessibility, transcription factor binding, and long-range interaction data. RESULTS: Clustering of the significant mimQTLs identified distinct groups of miRNAs and CpGs that reflect important biological processes associated with breast cancer pathogenesis. Notably, two major miRNA clusters were related to immune or fibroblast infiltration, hence identifying miRNAs associated with cells of the tumor microenvironment, while another large cluster was related to estrogen receptor (ER) signaling. Studying the chromatin landscape surrounding CpGs associated with the estrogen signaling cluster, we found that miRNAs from this cluster are likely to be regulated through DNA methylation of enhancers bound by FOXA1, GATA2, and ER-alpha. Further, at the hub of the estrogen cluster, we identified hsa-miR-29c-5p as negatively correlated with the mRNA and protein expression of DNA methyltransferase DNMT3A, a key enzyme regulating DNA methylation. We found deregulation of hsa-miR-29c-5p already present in pre-invasive breast lesions and postulate that hsa-miR-29c-5p may trigger early event abnormal DNA methylation in ER-positive breast cancer. CONCLUSIONS: We describe how miRNA expression and DNA methylation interact and associate with distinct breast cancer phenotypes.

The expression of the long NEAT1_2 isoform is associated with human epidermal growth factor receptor 2-positive breast cancers.

The long non-coding RNA NEAT1 locus is transcribed into two overlapping isoforms, NEAT1_1 and NEAT1_2, of which the latter is essential for the assembly of nuclear paraspeckles. NEAT1 is abnormally expressed in a wide variety of human cancers. Emerging evidence suggests that the two isoforms have distinct functions in gene expression regulation, and recently it was shown that NEAT1_2, but not NEAT1_1, expression predicts poor clinical outcome in cancer. Here, we report that NEAT1_2 expression correlates with HER2-positive breast cancers and high-grade disease. We provide evidence that NEAT1_1 and NEAT1_2 have distinct expression pattern among different intrinsic breast cancer subtypes. Finally, we show that NEAT1_2 expression and paraspeckle formation increase upon lactation in humans, confirming what has previously been demonstrated in mice.

Widespread alternative exon usage in clinically distinct subtypes of Invasive Ductal Carcinoma.

Cancer cells can have different patterns of exon usage of individual genes when compared to normal tissue, suggesting that alternative splicing may play a role in shaping the tumor phenotype. The discovery and identification of gene variants has increased dramatically with the introduction of RNA-sequencing technology, which enables whole transcriptome analysis of known, as well as novel isoforms. Here we report alternative splicing and transcriptional events among subtypes of invasive ductal carcinoma in The Cancer Genome Atlas (TCGA) Breast Invasive Carcinoma (BRCA) cohort. Alternative exon usage was widespread, and although common events were shared among three subtypes, ER+ HER2-, ER- HER2-, and HER2+, many events on the exon level were subtype specific. Additional RNA-seq analysis was carried out in an independent cohort of 43 ER+ HER2- and ER- HER2- primary breast tumors, confirming many of the exon events identified in the TCGA cohort. Alternative splicing and transcriptional events detected in five genes, MYO6, EPB41L1, TPD52, IQCG, and ACOX2 were validated by qRT-PCR in a third cohort of 40 ER+ HER2- and ER- HER2- patients, showing that these events were truly subtype specific.

Expression of an estrogen-regulated variant transcript of the peroxisomal branched chain fatty acid oxidase ACOX2 in breast carcinomas.

BACKGROUND: Alternate transcripts from a single gene locus greatly enhance the combinatorial flexibility of the human transcriptome. Different patterns of exon usage have been observed when comparing normal tissue to cancers, suggesting that variant transcripts may play a role in the tumor phenotype. METHODS: Ribonucleic acid-sequencing (RNA-seq) data from breast cancer samples was used to identify an intronic start variant transcript of Acyl-CoA oxidase 2, ACOX2 (ACOX2-i9). Difference in expression between Estrogen Receptor (ER) positive and ER negative patients was assessed by the Wilcoxon rank sum test, and the findings validated in The Cancer Genome Atlas (TCGA) breast cancer dataset (BRCA). ACOX2-i9 expression was also assessed in cell lines using both quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blot analysis. Knock down by short hairpin RNA (shRNA) and colony formation assays were used to determine whether ACOX2-i9 expression would influence cellular fitness. The effect of ACOX2-i9 expression on patient survival was assessed by the Kaplan-Meier survival function, and association to clinical parameters was analyzed using a Fisher exact test. RESULTS: The expression and translation of ACOX2-i9 into a 25 kDa protein was demonstrated in HepG2 cells as well as in several breast cancer cell lines. shRNA knock down of the ACOX2-i9 variant resulted in decreased cell viability of T47D and MDA-MB 436 cells. Moreover, expression of ACOX2-i9 was shown to be estrogen regulated, being induced by propyl pyrazoletriol and inhibited by tamoxifen and fulvestrant in ER+ T47D and Mcf-7 cells, but not in the ER- MDA-MB 436 cell line. This variant transcript showed expression predominantly in ER-positive breast tumors as assessed in our initial set of 53 breast cancers and further validated in 87 tumor/normal pairs from the TCGA breast cancer dataset, and expression was associated with better outcome in ER positive patients. CONCLUSIONS: ACOX2-i9 is specifically enriched in ER+ breast cancers where expression of the variant is associated with improved outcome. These data identify variant ACOX2 as a potential novel therapeutic biomarker in ER+ breast tumors.

Ubiquitination of fibroblast growth factor receptor 1 is required for its intracellular sorting but not for its endocytosis.

Endocytosis and targeting of growth factor receptors for lysosomal degradation have been associated with ubiquitination of the intracellular part of the receptors. To elucidate the role of receptor ubiquitination in internalization and sorting of fibroblast growth factor receptor (FGFR), we constructed several mutants of FGFR1 in which lysines, potential ubiquitination sites, were substituted for arginines. Substitution of all lysine residues in the intracellular part of FGFR1 resulted in inactivation of the tyrosine kinase domain of the receptor. However, several multilysine FGFR1 mutants, where up to 26 of 29 lysines in the intracellular part of the receptor were mutated, retained tyrosine kinase activity. The active multilysine mutants were poorly ubiquitinated, but internalized normally, indicating that ubiquitination of the receptor is not required for endocytosis. In contrast, degradation of the multilysine mutants was dramatically reduced as the mutants were inefficiently transported to lysosomes but rather sorted to recycling endosomes. The altered sorting resulted in sustained signaling. The duration of FGFR1 signaling seems to be tightly regulated by receptor ubiquitination and subsequent sorting to the lysosomes for degradation.