Edited by: Hongwei Wang, Sun Yat-sen University, China
Reviewed by: Hao Cai, First Affiliated Hospital of Gannan Medical University, China; Haidan Yan, Fujian Medical University, China
This article was submitted to Computational Genomics, a section of the journal Frontiers in Genetics
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Metastasis is a complex process that involved in various genetic and epigenetic alterations during the progression of breast cancer. Recent evidences have indicated that the mutation in the genome sequence may not be the key factor for increasing metastatic potential. Epigenetic changes were revealed to be important for metastatic phenotypes transition with the development in understanding the epigenetic basis of breast cancer. Herein, we aim to present the potential epigenetic drivers that induce dysregulation of genes related to breast tumor growth and metastasis, with a particular focus on histone modification including histone acetylation and methylation. The pervasive role of major histone modification enzymes in cancer metastasis such as histone acetyltransferases (HAT), histone deacetylases (HDACs), DNA methyltransferases (DNMTs), and so on are demonstrated and further discussed. In addition, we summarize the recent advances of next-generation sequencing technologies and microfluidic-based devices for enhancing the study of epigenomic landscapes of breast cancer. This feature also introduces several important biotechnologists for identifying robust epigenetic biomarkers and enabling the translation of epigenetic analyses to the clinic. In summary, a comprehensive understanding of epigenetic determinants in metastasis will offer new insights of breast cancer progression and can be achieved in the near future with the development of innovative epigenomic mapping tools.
Breast cancer is the most common cancer in women, impacting 2.1 million women each year based on the data from WHO. Furthermore, breast cancer also causes the greatest number of cancer-related deaths (
For a long time, breast cancer is considered to be a disease of the genome, predominantly resulting from mutations in key genes such as
DNA methylation is defined as the covalent bonding of a methyl group to the cytosine of the genomic CpG dinucleotide by two types of DNA methyltransferases (DNMTs), which causes changes in chromatin structure, DNA conformation, DNA stability and the way in which DNA interacts with proteins, thereby controlling gene expression (
Overview of breast cancer related-epigenetic modifications.
Another key driver in epigenetics is histone modification, which modulates the structure of the chromatin, thereby altering the accessibility of DNA. And the main histone modifications including enzymes such as histone acetyltransferases (HATs), histone deacetylases (HDACs) and histone methyltransferases (HMTs). The acetyl groups or the methyl groups are added to the amino acid tails of the histone proteins when most of the histone modifications occur (
As we know, DNA methylation and histone modification influence each other during nucleosome remodeling and gene expression regulation, which might impact the development of the cellular processes (
In addition, another epigenetic regulatory mechanism is miRNAs, which was found to play an important role in tumor development in breast cancer. The main function of miRNA is to induce the degradation of targeted mRNA or inhibit the translation of targeted mRNA. These miRNAs can generally inhibit the expression of multiple genes and participate in the regulation of cell proliferation and differentiation. During the progression of breast cancer, the regulation of miRNA expression will lead to an imbalance in the cellular level of miRNA, and ultimately worsen the disease (
As mentioned above, a large amount of literature has shown that epigenetics is one of the main players in breast cancer metastasis. Therefore, it is currently important to identify the role of these epigenetics in tumor metastasis and how to better analyze these epigenetic drivers related to tumor metastasis. We mainly focus on histone modification events as histone modification contributes to cancer metastasis by controlling the transition of different metastatic phenotypes in breast cancer cells.
The histone modifications are proposed to constitute a “histone code” to maintain histone interactions with chromatin-associated proteins and therefore allow the regulation of specific downstream function. Accordingly, HATs as the “writer” enzymes could transfer acetyl groups to the particular groups of lysine or arginine residues in histone tails, which results in gene activation. In contrast, HDAC as the “eraser” enzymes could remove the acetyl groups from the tail of the histones and repress the target gene. Both “writer” and “eraser” enzymes modify histones could control the active or silent states of chromatin, which can transcriptionally regulate the transcription of genetic information encoded in DNA. Thus researchers use this to develop drugs for clinical diagnosis and treatment and provide corresponding therapeutic targets. These drugs can effectively block breast cancer metastasis and tumor progression via the impact on histone modification, especially for histone acetylation (
A proposed role of histone modification in the transcriptional regulation of genes involved in breast cancer metastasis and assign functional significance to these epigenetic drivers.
From a theoretical point of view, histone acetylation will reduce the positive charge of histones, thereby reducing the binding of nucleosomes and DNA, activating gene expression. Generally speaking, the degree of histone acetylation will be higher in the promoter region of active genes, which can affect the initiation of gene transcription and prolong gene transcription. Furthermore, histone acetylation adjusts the structure of chromatin, which in turn changes the transcriptional activity of genes. For example, the structure of acetylated chromatin becomes loose, which is related to activated transcription. The deacetylated chromatin becomes concentrated and supercoiled and is associated with transcriptional inhibition (in the case of breast cancer, it inhibits the expression of tumor suppressor genes). The acetylation reaction of histones is controlled by HATs and HADC. The reaction is a fast and reversible process. HATs are subdivided into four classes: GNAT, p300/CBP, MYST, and fungal Rtt109 family based on the catalytic mechanisms and sequence homology (
Another major category related to histone acetylation is HDACs. HDACs can be divided into 4 groups: class I (HDACs 1, 2, 3, and 8), class II (HDACs 4, 5, 6, 7, 9, and 10), class III (SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7), and class IV (HDAC11) according to their sequence homology, subcellular location, and the features of the catalytic site (
Histone methylation is a covalent modification that occurs on arginine and lysine. Arginine can be monomethylated or dimethylated, and lysine can be monomethylated, dimethylated or trimethylated. Similar to DNA methylation, the process of histone methylation involves the transfer of methyl groups from S-adenosylmethionine (SAM) to lysine or arginine residues by HMT, and histone demethylase (HDM) removes methyl groups from the histone. The expression of HMTs might impact on the progression of tumor and metastasis. For example, PRMT1, which is a targeted HMT, could bind to the promoter of ZEB1 and mediates histone methylation to induce the EMT process in breast cancer cells (
The phosphorylation modification of histones H2B and H3 was demonstrated to play an important role in DNA repair, mitosis and gene regulation (
As we know, the classification of breast tumors is based on their hormone receptor status and pathologic features and the histone modifications play important roles in the regulation of gene expression in cancer pathogenesis. Thus there might be a subtype-specific regulation in breast cancer which is related to histone modifications. Recently, efforts have been taken to investigate the association between histone modifications and the subtypes of metastatic breast cancer (
With the efforts to advanced therapeutics in the treatment of breast cancer, yet breast cancer metastasis remains the leading cause of death in women patients. The major reason is that the epigenome effects in breast cancer metastasis were still unclear. Currently, available technologies allow the study the epigenetic drivers in breast cancer metastasis, however, these traditional tools limited the accuracy and precision in the investigation of epigenomic signatures, which might enlarge the gaps between promise and realization of epigenomic therapy. Therefore, concerns should be taken to develop novel approaches to better describe the epigenome in breast cancer metastasis. The following sections here purposed the current new technologies for profiling epigenetic determinants of breast cancer metastasis, including the next-generation sequencing (NGS) and microfluidic platform for mapping the epigenome recently.
For many years, the research of breast cancer epigenetics has been only aimed at the DNA methylation analysis of a specific gene and the identification of histone modifications. These research results have laid the foundation for the study of breast cancer epigenetics, and revealed that the epigenetic information can be used as a new generation of clinical diagnostic markers and new targets for anti-tumor therapy. The technological development of NGS in recent years has promoted the progress in the study of breast cancer epigenetics gradually from a single gene to a genome-wide scale. Recently, whole genome bisulfite sequencing (WGBS) is the gold standard for DNA methylation research (
Overview of the major epigenetic modification detection methods.
Methods based on NGS are also extensively used to study histone modification in recent years. Chromatin immunoprecipitation-deep sequencing (ChIP-Seq) is a high-throughput technology that combines chromatin immunoprecipitation (ChIP) with deep sequencing technology (
Deep RNA sequencing (RNA-seq) has been used to reveal the roles miRNAs play in epigenetic regulation in recent years. Among many NGS technologies, the first method used for miRNA detection is the second-generation methods including Roche’s 454 pyrosequencing and Illumina (Solexa) sequencing (
In addition to NGS, microfluidics provides efficient platforms to maintain epigenomes study on DNA methylation, histone modifications and 3D chromatin structures (
Bisulfite conversation-based detection is widely used in DNA methylation analysis in recent years. As a high-throughput and automated device, the microfluidic platform allows bisulfite treatment with higher efficiency when compared with traditional methods. The on-chip bisulfite conversion was processed in a microfluidic chip which was integrated with the MS-PCR analysis unit, the device has achieved the methylated DNA detection within 80 min with low-input sample (
Chromatin immunoprecipitation is used as the gold standard to analyze histone modifications, however, limitations of ChIP including large sample size, low throughput, and poor robustness prevent its routine implementation. Recent efforts have been taken to improve ChIP assay with microfluidic tools by reducing the reagent volume, maintaining multiple samples parallelly and process workflow automated (
Different from the analysis methods for DNA methylation and histone modification, miRNA detection is mainly based on RNA-sequencing technologies. However, many microfluidic chips have tried to measure miRNA in recent years without RNA-sequencing. For example, a SERS-based microfluidic device was used to detect miR-222, which is involved in multiple cancers, with silver-coated porous silicon membranes (
Increasing evidence shows that epigenomic can indicate the progression of breast cancer. The epigenomic profiles including DNA methylation and histone modification and so on may lead to abnormal protein expression in signaling pathways related to tumor metastasis, and finally cause tumor progression. We demonstrated that epigenetics plays a key role in breast cancer metastasis, but it also leaves many important open questions. For example, because the tumor microenvironment also plays an important role in breast cancer metastasis, whether the tumor microenvironment is also changed by the epigenetic changes of tumor cells. Then, since EMT is involved in multiple processes of breast cancer metastasis, it is usually temporarily activated or partially activated in breast cancer cells. Which histone modification enzymes such as the SIRT family play an important role in it? What signaling pathways do these upstream regulatory genes use to affect downstream gene expression and affect the balance between EMT cell state at last? In addition, we have described that there are a variety of enzymes involved in the change of epigenetics, however, the analysis methods for proteins or enzymes are still lacking. The analysis of these enzymes including DNMT, HMT, HDAC, etc., lacks high-throughput analysis methods. Finally, is there any relationship between epigenetic instability and the heterogeneity of tumor cells? Does the reversibility of such epigenetics such as methylation and histone modification increase tumor heterogeneity and affect tumor metastasis.
By analyzing the relationship between epigenetic information and breast cancer metastasis, the epigenome mechanism of breast cancer metastasis can be clarified, so as to provide insights for diagnosis and treatment. There are many new methods including NGS and microfluidics that can be used to detect epigenetic information such as DNA methylation and histone modification, but from the current progress, the work of single-cell epigenome analysis is less than that of single-cell genome and single-cell transcriptomics. Therefore, the powerful analysis capabilities of NGS and the miniaturization and integration characteristics of microfluidic chips should be combined to promote the investigation of single-cell epigenetics of breast cancer metastasis. Secondly, in the future, epigenetics research will also develop in a direction that is more suitable for early clinical screening. For example, to reduce the number of testing samples, improve the high-throughput analysis capabilities of the device, and offer automatic process, the development of novel microfluidic chips makes the analysis of these epigenetic biomarkers as easy and simple as clinical biochemical analysis. Finally, these methods will need to be validated using clinical samples (such as tissue biopsies) and compared with batch methods to ensure accurate data coverage before they can be reliably applied in the clinic.
All in all, the progress on epigenetics in breast cancer metastasis helps to better understand the molecular mechanisms associated with metastasis, thereby helping to accelerate the development of new metastatic treatment strategies and biomarkers.
JZ and NX contributed to the conception of the manuscript. JZ wrote the manuscript. QH and FY revised the manuscript. All authors gave approval to the final version of the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.