Philippe Gascard

The clonal and mutational evolution spectrum of primary triple-negative breast cancers.

Primary triple-negative breast cancers (TNBCs), a tumour type defined by lack of oestrogen receptor, progesterone receptor and ERBB2 gene amplification, represent approximately 16% of all breast cancers. Here we show in 104 TNBC cases that at the time of diagnosis these cancers exhibit a wide and continuous spectrum of genomic evolution, with some having only a handful of coding somatic aberrations in a few pathways, whereas others contain hundreds of coding somatic mutations. High-throughput RNA sequencing (RNA-seq) revealed that only approximately 36% of mutations are expressed. Using deep re-sequencing measurements of allelic abundance for 2,414 somatic mutations, we determine for the first time—to our knowledge—in an epithelial tumour subtype, the relative abundance of clonal frequencies among cases representative of the population. We show that TNBCs vary widely in their clonal frequencies at the time of diagnosis, with the basal subtype of TNBC showing more variation than non-basal TNBC. Although p53 (also known as TP53), PIK3CA and PTEN somatic mutations seem to be clonally dominant compared to other genes, in some tumours their clonal frequencies are incompatible with founder status. Mutations in cytoskeletal, cell shape and motility proteins occurred at lower clonal frequencies, suggesting that they occurred later during tumour progression. Taken together, our results show that understanding the biology and therapeutic responses of patients with TNBC will require the determination of individual tumour clonal genotypes.

DNA hypomethylation within specific transposable element families associates with tissue-specific enhancer landscape

Transposable element (TE)-derived sequences comprise half of the human genome and DNA methylome and are presumed to be densely methylated and inactive. Examination of genome-wide DNA methylation status within 928 TE subfamilies in human embryonic and adult tissues identified unexpected tissue-specific and subfamily-specific hypomethylation signatures. Genes proximal to tissue-specific hypomethylated TE sequences were enriched for functions important for the relevant tissue type, and their expression correlated strongly with hypomethylation within the TEs. When hypomethylated, these TE sequences gained tissue-specific enhancer marks, including monomethylation of histone H3 at lysine 4 (H3K4me1) and occupancy by p300, and a majority exhibited enhancer activity in reporter gene assays. Many such TEs also harbored binding sites for transcription factors that are important for tissue-specific functions and showed evidence of evolutionary selection. These data suggest that sequences derived from TEs may be responsible for wiring tissue type–specific regulatory networks and may have acquired tissue-specific epigenetic regulation.

Functional DNA methylation differences between tissues, cell types, and across individuals discovered using the M&M algorithm

DNA methylation plays key roles in diverse biological processes such as X chromosome inactivation, transposable element repression, genomic imprinting, and tissue-specific gene expression. Sequencing-based DNA methylation profiling provides an unprecedented opportunity to map and compare complete DNA methylomes. This includes one of the most widely applied technologies for measuring DNA methylation: methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq), coupled with a complementary method, methylation-sensitive restriction enzyme sequencing (MRE-seq). A computational approach that integrates data from these two different but complementary assays and predicts methylation differences between samples has been unavailable. Here, we present a novel integrative statistical framework M&M (for integration of MeDIP-seq and MRE-seq) that dynamically scales, normalizes, and combines MeDIP-seq and MRE-seq data to detect differentially methylated regions. Using sample-matched whole-genome bisulfite sequencing (WGBS) as a gold standard, we demonstrate superior accuracy and reproducibility of M&M compared to existing analytical methods for MeDIP-seq data alone. M&M leverages the complementary nature of MeDIP-seq and MRE-seq data to allow rapid comparative analysis between whole methylomes at a fraction of the cost of WGBS. Comprehensive analysis of nineteen human DNA methylomes with M&M reveals distinct DNA methylation patterns among different tissue types, cell types, and individuals, potentially underscoring divergent epigenetic regulation at different scales of phenotypic diversity. We find that differential DNA methylation at enhancer elements, with concurrent changes in histone modifications and transcription factor binding, is common at the cell, tissue, and individual levels, whereas promoter methylation is more prominent in reinforcing fundamental tissue identities.

Carcinoma-associated fibroblasts: orchestrating the composition of malignancy

The tumor stroma is no longer seen solely as physical support for mutated epithelial cells but as an important modulator and even a driver of tumorigenicity. Within the tumor stromal milieu, heterogeneous populations of fibroblast-like cells, collectively termed carcinoma-associated fibroblasts (CAFs), are key players in the multicellular, stromal-dependent alterations that contribute to malignant initiation and progression. This review focuses on novel insights into the contributions of CAFs to disease progression, emergent events leading to the generation of CAFs, identification of CAF-specific biomarkers predictive of disease outcome, and recent therapeutic approaches aimed at blunting or reverting detrimental protumorigenic phenotypes associated with CAFs.

Rare somatic cells from human breast tissue exhibit extensive lineage plasticity

We identified cell surface markers associated with repression of p16INK4a/cyclin-dependent kinase inhibitor 2A(CDKN2A), a critical determinant in the acquisition of a plastic state. These cell surface markers allowed direct isolation of rare cells from healthy human breast tissue that exhibit extensive lineage plasticity. This subpopulation is poised to transcribe plasticity markers, OCT3/4, SOX2, and NANOG, at levels similar to those measured in human embryonic stem cells and to acquire a plastic state sensitive to environmental programming. In vitro, in vivo, and teratoma assays demonstrated that either a directly sorted (uncultured) or a single-cell (clonogenic) cell population from primary tissue can differentiate into functional derivatives of each germ layer, ectodermal, endodermal, and mesodermal. In contrast to other cells that express OCT3/4, SOX2, and NANOG, these human endogenous plastic somatic cells are mortal, express low telomerase activity, expand for an extensive but finite number of population doublings, and maintain a diploid karyotype before arresting in G1.

Intermediate DNA methylation is a conserved signature of genome regulation

The role of intermediate methylation states in DNA is unclear. Here, to comprehensively identify regions of intermediate methylation and their quantitative relationship with gene activity, we apply integrative and comparative epigenomics to 25 human primary cell and tissue samples. We report 18,452 intermediate methylation regions located near 36% of genes and enriched at enhancers, exons and DNase I hypersensitivity sites. Intermediate methylation regions average 57% methylation, are predominantly allele-independent and are conserved across individuals and between mouse and human, suggesting a conserved function. These regions have an intermediate level of active chromatin marks and their associated genes have intermediate transcriptional activity. Exonic intermediate methylation correlates with exon inclusion at a level between that of fully methylated and unmethylated exons, highlighting gene context-dependent functions. We conclude that intermediate DNA methylation is a conserved signature of gene regulation and exon usage.

Epigenetic and transcriptional determinants of the human breast

While significant effort has been dedicated to the characterization of epigenetic changes associated with prenatal differentiation, relatively little is known about the epigenetic changes that accompany post-natal differentiation where fully functional differentiated cell types with limited lifespans arise. Here we sought to address this gap by generating epigenomic and transcriptional profiles from primary human breast cell types isolated from disease-free human subjects. From these data we define a comprehensive human breast transcriptional network, including a set of myoepithelial- and luminal epithelial-specific intronic retention events. Intersection of epigenetic states with RNA expression from distinct breast epithelium lineages demonstrates that mCpG provides a stable record of exonic and intronic usage, whereas H3K36me3 is dynamic. We find a striking asymmetry in epigenomic reprogramming between luminal and myoepithelial cell types, with the genomes of luminal cells harbouring more than twice the number of hypomethylated enhancer elements compared with myoepithelial cells.

Coordinate Transcriptional and Translational Repression of p53 by TGF-β1 Impairs the Stress Response

Cellular stress results in profound changes in RNA and protein synthesis. How cells integrate this intrinsic, p53-centered program with extracellular signals is largely unknown. We demonstrate that TGF-β1 signaling interferes with the stress response through coordinate transcriptional and translational repression of p53 levels, which reduces p53-activated transcription, and apoptosis in precancerous cells. Mechanistically, E2F-4 binds constitutively to the TP53 gene and induces transcription. TGF-β1-activated Smads are recruited to a composite Smad/E2F-4 element by an E2F-4/p107 complex that switches to a Smad corepressor, which represses TP53 transcription. TGF-β1 also causes dissociation of ribosomal protein RPL26 and elongation factor eEF1A from p53 mRNA, thereby reducing p53 mRNA association with polyribosomes and p53 translation. TGF-β1 signaling is dominant over stress-induced transcription and translation of p53 and prevents stress-imposed downregulation of Smad proteins. Thus, crosstalk between the TGF-β and p53 pathways defines a major node of regulation in the cellular stress response, enhancing drug resistance.

Regulatory network decoded from epigenomes of surface ectoderm-derived cell types.

Developmental history shapes the epigenome and biological function of differentiated cells. Epigenomic patterns have been broadly attributed to the three embryonic germ layers. Here we investigate how developmental origin influences epigenomes. We compare key epigenomes of cell types derived from surface ectoderm (SE), including keratinocytes and breast luminal and myoepithelial cells, against neural crest-derived melanocytes and mesoderm-derived dermal fibroblasts to identify SE differentially methylated regions (SE-DMRs). DNA methylomes of neonatal keratinocytes share many more DMRs with adult breast luminal and myoepithelial cells than with melanocytes and fibroblasts from the same neonatal skin. This suggests that SE origin contributes to DNA methylation patterning, while shared skin tissue environment has limited effect on epidermal keratinocytes. Hypomethylated SE-DMRs are in proximity to genes with SE relevant functions. They are also enriched for enhancer- and promoter-associated histone modifications in SE-derived cells, and for binding motifs of transcription factors important in keratinocyte and mammary gland biology. Thus, epigenomic analysis of cell types with common developmental origin reveals an epigenetic signature that underlies a shared gene regulatory network.

Similarities and differences in the structure and function of 4.1G and 4.1R135, two protein 4.1 paralogues expressed in erythroid cells.

Membrane skeletal protein 4.1R is the prototypical member of a family of four highly paralogous proteins that include 4.1G, 4.1N and 4.1B. Two isoforms of 4.1R (4.1R135 and 4.1R80), as well as 4.1G, are expressed in erythroblasts during terminal differentiation, but only 4.1R80 is present in mature erythrocytes. Although the function of 4.1R isoforms in erythroid cells has been well characterized, there is little or no information on the function of 4.1G in these cells. In the present study, we performed detailed characterization of the interaction of 4.1G with various erythroid membrane proteins and the regulation of these interactions by calcium-saturated calmodulin. Like both isoforms of 4.1R, 4.1G bound to band 3, glycophorin C, CD44, p55 and calmodulin. While both 4.1G and 4.1R135 interact with similar affinity with CD44 and p55, there are significant differences in the affinity of their interaction with band 3 and glycophorin C. This difference in affinity is related to the non-conserved N-terminal headpiece region of the two proteins that is upstream of the 30 kDa membrane-binding domain that harbours the binding sites for the various membrane proteins. The headpiece region of 4.1G also contains a high-affinity calcium-dependent calmodulin-binding site that plays a key role in modulating its interaction with various membrane proteins. We suggest that expression of the two paralogues of protein 4.1 with different affinities for band 3 and glycophorin C is likely to play a role in assembly of these two membrane proteins during terminal erythroid differentiation.