Antoni Hurtado

Switch in FOXA1 status associates with endometrial cancer progression

Background The transcription factor Forkhead box A1 (FOXA1) is suggested to be important in hormone dependent cancers, although with little data for endometrial cancer. We investigated expression levels of FOXA1 in primary and metastatic endometrial cancer in relation to clinical phenotype, and transcriptional alterations related to FOXA1 status. Methods Protein expression of FOXA1 was explored by immunohistochemistry in 529 primary and 199 metastatic endometrial carcinoma lesions. mRNA levels from corresponding 158 fresh frozen primary and 42 metastatic lesions were analyzed using Agilent Microarrays (44k) in parallel. Results Low FOXA1 protein expression in primary tumors significantly correlated with low FOXA1 mRNA, high age, non-endometrioid histology, high grade, loss of ERα and PR and poor survival (all p-values <0.05). Through a Connectivity Map search, HDAC inhibitors were suggested as potential treatment for patients with low FOXA1 expression. An increase in FOXA1 expression was observed from primary to metastatic lesions and it correlated with CDKN2A expression in metastases. Conclusion Low FOXA1 is associated with poor survival and suggests a potential for HDAC inhibitors in endometrial carcinoma. A switch in FOXA1 expression from primary to metastatic lesions is observed and gene expression indicates a link between FOXA1 and CDKN2A in metastatic lesions.

The 5p12 breast cancer susceptibility locus affects MRPS30 expression in estrogen-receptor positive tumors

Genome‐wide association studies have identified numerous loci linked to breast cancer susceptibility, but the mechanism by which variations at these loci influence susceptibility is usually unknown. Some variants are only associated with particular clinical subtypes of breast cancer. Understanding how and why these variants influence subtype‐specific cancer risk contributes to our understanding of cancer etiology. We conducted a genome‐wide expression Quantitative Trait Locus (eQTL) study in a discovery set of 287 breast tumors and 97 normal mammary tissue samples and a replication set of 235 breast tumors. We found that the risk‐associated allele of rs7716600 in the 5p12 estrogen receptor‐positive (ER‐positive) susceptibility locus was associated with elevated expression of the nearby gene MRPS30 exclusively in ER‐positive tumors. We replicated this finding in 235 independent tumors. Further, we showed the rs7716600 risk genotype was associated with decreased MRPS30 promoter methylation exclusively in ER‐positive breast tumors. In vitro studies in MCF‐7 cells carrying the protective genotype showed that estrogen stimulation decreased MRPS30 promoter chromatin availability and mRNA levels. In contrast, in 600MPE cells carrying the risk genotype, estrogen increased MRPS30 expression and did not affect promoter availability. Our data suggest the 5p12 risk allele affects MRPS30 expression in estrogen‐responsive tumor cells after tumor initiation by a mechanism affecting chromatin availability. These studies emphasize that the genetic architecture of breast cancer is context‐specific, and integrated analysis of gene expression and chromatin remodeling in normal and tumor tissues will be required to explain the mechanisms of risk alleles.

CTCF modulates Estrogen Receptor function through specific chromatin and nuclear matrix interactions

Enhancer regions and transcription start sites of estrogen-target regulated genes are connected by means of Estrogen Receptor long-range chromatin interactions. Yet, the complete molecular mechanisms controlling the transcriptional output of engaged enhancers and subsequent activation of coding genes remain elusive. Here, we report that CTCF binding to enhancer RNAs is enriched when breast cancer cells are stimulated with estrogen. CTCF binding to enhancer regions results in modulation of estrogen-induced gene transcription by preventing Estrogen Receptor chromatin binding and by hindering the formation of additional enhancer-promoter ER looping. Furthermore, the depletion of CTCF facilitates the expression of target genes associated with cell division and increases the rate of breast cancer cell proliferation. We have also uncovered a genomic network connecting loci enriched in cell cycle regulator genes to nuclear lamina that mediates the CTCF function. The nuclear lamina and chromatin interactions are regulated by estrogen-ER. We have observed that the chromatin loops formed when cells are treated with estrogen establish contacts with the nuclear lamina. Once there, the portion of CTCF associated with the nuclear lamina interacts with enhancer regions, limiting the formation of ER loops and the induction of genes present in the loop. Collectively, our results reveal an important, unanticipated interplay between CTCF and nuclear lamina to control the transcription of ER target genes, which has great implications in the rate of growth of breast cancer cells.

Cooperating transcription factors mediate the function of estrogen receptor

Estrogen receptor (ER) is a hormone-regulated transcription factor that controls cell division and differentiation in the ovary, breast, and uterus. The expression of ER is a common feature of the majority of breast cancers, which is used as a therapeutic target. Recent genetic studies have shown that ER binding occurs in regions distant to the promoters of estrogen target genes. These studies have also demonstrated that ER binding is accompanied with the binding of other transcription factors, which regulate the function of ER and response to anti-estrogen therapies. In this review, we explain how these factors influence the interaction of ER to chromatin and their cooperation for ER transcriptional activity. Moreover, we describe how the expression of these factors dictates the response to anti-estrogen therapies. Finally, we discuss how cytoplasmatic signaling pathways may modulate the function of ER and its cooperating transcription factors.

Functional Genomic Methods to Study Estrogen Receptor Activity

Estrogen Receptor (ER) is a nuclear receptor that mediates the actions of estrogen and tamoxifen. ER is expressed in a major fraction of human breast cancers. Recently, genomic maps for estrogen- and tamoxifen-ER have been published. Interestingly, estrogen and tamoxifen induce similar genomic interactions and both ligands have been shown to use co-operating factors. The interactions of these co-operating factors within ER regions have impact both on ER-DNA interactions and gene expression regulated by estrogen and tamoxifen. Moreover, the study of chromatin changes induced by these factors has also provided significant insight into our understanding of ER transcriptional regulation. This methods review describes some functional genomic methods to study the influence of both ER ligands and ER co-operating factors. The analysis of protein-DNA interactions and chromatin changes can be explored by using classical and novel methods such as Chromatin Immunoprecipitation (ChIP) or Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE). This review also explores the properties of each of these methods and the advantages of combining them with high throughput sequencing.

DNA methylation at enhancers identifies distinct breast cancer lineages

Breast cancers exhibit genome-wide aberrant DNA methylation patterns. To investigate how these affect the transcriptome and which changes are linked to transformation or progression, we apply genome-wide expression–methylation quantitative trait loci (emQTL) analysis between DNA methylation and gene expression. On a whole genome scale, in cis and in trans, DNA methylation and gene expression have remarkably and reproducibly conserved patterns of association in three breast cancer cohorts (n = 104, n = 253 and n = 277). The expression–methylation quantitative trait loci associations form two main clusters; one relates to tumor infiltrating immune cell signatures and the other to estrogen receptor signaling. In the estrogen related cluster, using ChromHMM segmentation and transcription factor chromatin immunoprecipitation sequencing data, we identify transcriptional networks regulated in a cell lineage-specific manner by DNA methylation at enhancers. These networks are strongly dominated by ERα, FOXA1 or GATA3 and their targets were functionally validated using knockdown by small interfering RNA or GRO-seq analysis after transcriptional stimulation with estrogen.Breast cancers are classified based on gene expression profiles but it is well known that DNA methylation patterns are also altered. Here the authors, by combining genome-wide DNA methylation and gene expression analysis, identify transcriptional networks regulated by DNA methylation at enhancers able to separate specific breast cancer subtypes.

Human SHBG mRNA Translation Is Modulated by Alternative 5′-Non-Coding Exons 1A and 1B

Background The human sex hormone-binding globulin (SHBG) gene comprises at least 6 different transcription units (TU-1, -1A, -1B, -1C, -1D and -1E), and is regulated by no less than 6 different promoters. The best characterized are TU-1 and TU-1A: TU-1 is responsible for producing plasma SHBG, while TU-1A is transcribed and translated in the testis. Transcription of the recently described TU-1B, -1C, and -1D has been demonstrated in human prostate tissue and prostate cancer cell lines, as well as in other human cell lines such as HeLa, HepG2, HeK 293, CW 9019 and imr 32. However, there are no reported data demonstrating their translation. In the present study, we aimed to determine whether TU-1A and TU-1B are indeed translated in the human prostate and whether 5′ UTR exons 1A and 1B differently regulate SHBG translation. Results Cis-regulatory elements that could potentially regulate translation were identified within the 5′UTRs of SHBG TU-1A and TU–1B. Although full-length SHBG TU-1A and TU-1B mRNAs were present in prostate cancer cell lines, the endogenous SHBG protein was not detected by western blot in any of them. LNCaP prostate cancer cells transfected with several SHBG constructs containing exons 2 to 8 but lacking the 5′UTR sequence did show SHBG translation, whereas inclusion of the 5′UTR sequences of either exon 1A or 1B caused a dramatic decrease in SHBG protein levels. The molecular weight of SHBG did not vary between cells transfected with constructs with or without the 5′UTR sequence, thus confirming that the first in-frame ATG of exon 2 is the translation start site of TU-1A and TU-1B. Conclusions The use of alternative SHBG first exons 1A and 1B differentially inhibits translation from the ATG situated in exon 2, which codes for methionine 30 of transcripts that begin with the exon 1 sequence.

HER2 expression patterns in paired primary and metastatic endometrial cancer lesions

Background:Despite successful implementation of drugs targeting the human epidermal growth factor receptor 2 (HER2) receptor in breast and gastric cancers, the potential of HER2 as a therapeutic target in other cancers has been less studied, including endometrial cancer. We investigated expression levels of HER2 (ERBB2) in a large cohort of endometrial cancer lesions, also including complex atypical hyperplasia and metastatic lesions.Methods:67 precursor lesions, 790 primary endometrial cancers and 383 metastatic lesions were investigated for HER2 expression in relation to clinicopathologic features and outcome. Protein levels were assessed by immunohistochemistry (using the HercepTest and staining index (SI) criteria), mRNA levels by microarrays and amplification status by chromogenic in situ hybridisation.Results:High HER2 protein levels were significantly associated with features of aggressive disease and increased mRNA ERBB2 levels. HER2 expression defined by the SI proved to be a better predictor of survival compared with the HercepTest. A discordant HER2 expression pattern between paired primary and metastatic lesions was detected, revealing substantial reduction in HER2 expression from primary to metastatic disease.Conclusions:Loss of HER2 expression is common in metastatic endometrial cancer lesions and assessment of HER2 levels in the metastatic lesions may be important to define the potential benefit of anti-HER2 treatments in endometrial cancer patients.

A functional link between FOXA1 and breast cancer SNPs

Genome-wide association studies have revealed a multitude of breast cancer-associated SNPs. The majority of these SNPs are located in noncoding regions of the genome. Yet how they contribute to breast cancer development is unknown. Recently, a groundbreaking study by the Lupien group has shown that risk-associated SNPs of breast cancer are enriched for FOXA1 binding sites, which influences the function of this transcription factor.

Modulation of gene transcription and epigenetics of colon carcinoma cells by bacterial membrane vesicles

Interactions between bacteria and colon cancer cells influence the transcription of the host cell. Yet is it undetermined whether the bacteria itself or the communication between the host and bacteria is responsible for the genomic changes in the eukaryotic cell. Now, we have investigated the genomic and epigenetic consequences of co-culturing colorectal carcinoma cells with membrane vesicles from pathogenic bacteria Vibrio cholerae and non-pathogenic commensal bacteria Escherichia coli. Our study reveals that membrane vesicles from pathogenic and commensal bacteria have a global impact on the gene expression of colon-carcinoma cells. The changes in gene expression correlate positively with both epigenetic changes and chromatin accessibility of promoters at transcription start sites of genes induced by both types of membrane vesicles. Moreover, we have demonstrated that membrane vesicles obtained only from V. cholerae induced the expression of genes associated with epithelial cell differentiation. Altogether, our study suggests that the observed genomic changes in host cells might be due to specific components of membrane vesicles and do not require communication by direct contact with the bacteria.