Breast cancer is one of the most common forms of cancer, and some subtypes are among the most difficult of all cancers to treat. The involvement of many different cells makes targeting such tumors challenging.

Salk Institute researchers now report on the use of a state-of-the-art technology known as single-nucleus (sn) assay for transposase-accessible chromatin (ATAC) sequencing (snATAC-seq) to profile how specific types of mammary cells differentiate during development. The findings, published in Cell Reports, provide new insights into normal breast development, and could ultimately point to new therapeutic strategies for different subtypes of breast cancer. The researchers have also made their findings available through a free online resource.

“In order to understand what goes wrong in breast cancer, we need to first understand how normal development works,” said research lead Geoffrey Wahl, PhD, a professor in the Gene Expression Laboratory. “This study represents a major step in that direction, as we were able to profile each cell during breast development. We expect this information to be a valuable hypothesis-generating resource for the mammary gland community.” The team’s work is published in a paper titled, “Single-Cell Chromatin Analysis of Mammary Gland Development Reveals Cell-State Transcriptional Regulators and Lineage Relationships.”

The specialized functions of different tissues result from the coordinated activities of diverse cell types that develop from progenitor cells, the authors explained. The process of cell differentiation into these specialized types is controlled through mechanisms including epigenetics. “The epigenetic programming of stem cells enables them to either retain their multi-potentiality or differentiate into the specific cell types.” Defining how epigenetic and molecular mechanisms are involved in orchestrating developmental plasticity and the differentiation of different cell types and their functions is important to help our understanding of processes that underlie tissue development and repair after injury, but also potentially how the activation of cancer genes can impact on these processes and drive cancer progression.

As the team pointed out, “The mammary gland is an excellent system for studying mechanisms of cellular specification because of its accessibility; the dramatic changes it undergoes in embryogenesis and postnatal development in response to puberty, pregnancy, and involution; and the substantial knowledge gained about factors involved in these cell-state transitions.”

Mature breast tissue contains two main cell types, which may be involved in breast cancer. Luminal cells line the ducts and produce milk, while the surrounding basal cells contract to move the milk through and out of the ducts. The Salk Institute scientists were interested in what drives the molecular changes that govern how, during development, stem cells become specialized into these types of cells. To investigate at the level of individual cells they used sn(ATAC)-seq profiling of both fetal and adult mammary cells to investigate how changes in DNA packaging into chromatin impact on whether certain genes are either accessible or inaccessible, which can then affect gene expression and the development of these different cell types.

“We sought to obtain a molecular map of these developing breast cells to better understand how breast tissues are formed during development and maintained during adulthood,” noted co-first author and staff scientist Christopher Dravis. “By examining differences in chromatin accessibility, we aimed to understand which regions of the genome affected transcription, the process that involves making RNA from DNA, and how that affected cell development,” added co-first author Zhibo Ma, PhD.

The researchers used the single-cell profiling technique to compare chromatin accessibility in adult mouse breast tissue with that in breast tissue at the late prenatal development stage. They separated the prenatal cells into groups with basal-like and luminal-like features, based on chromatin accessibility. The results, surprisingly, suggested that even before birth the individual cells were already “poised” to become either a basal cell or luminal cell. “… fetal cells at this stage of mammary development are starting to acquire adult-like chromatin accessibility, but they still largely possess their fetal-specific features,” the scientists commented. The findings suggested that most cells at the late prenatal stage are “weakly committed and biased toward either a luminal or basal fate,” and that “this likely positions these cells to differentiate rapidly into the respective cell type in response to appropriate microenvironmental cues after birth.” It is then possible that abnormal changes to these processes may lead to tumor development later on.

These are luminal cells (green) and basal cells (red) in mature mouse breast tissue. [Salk Institute]
The team also used bioinformatics and machine learning techniques to analyze different developing cell features, which highlighted a complex picture of breast development and maturation that could help research teams better understand the mechanisms that control breast tissue. “The data and strategies described provide a resource for future epigenetic studies of mammary cell regulation, a catalog of upstream control elements containing binding sites for cell-state-determining transcription factors, computational approaches that provide finer distinction of mammary cell states, and a pseudo time progression of mammary differentiation,” the investigators stated.

The Salk scientists have integrated their findings into a free online database, detailed in their paper, to help inform continuing research into cell growth, gene regulation, and other factors across multiple cell types and developmental states. The authors believe the database—which allows for comparison between chromatin accessibility and gene expression during normal breast development, among other components—could be used to improve our understanding of how breast cells become cancerous. “Our chromatin profiling of individual mammary cells at embryonic and adult developmental stages, and accompanying analyses that predict transcription factor activity and gene accessibility in relation to distinct mammary cell states, provide valuable resources to discover and validate cell-state regulators,” they wrote. “The links between mammary development and breast cancer suggest that this resource, which we have made available as a web-based app, will have significant utility in target discovery for breast cancers.”

“My objective has always been to help people with cancer and, by disseminating research as widely and as quickly as possible, we hope to accelerate research advancement and therapy development,” said Wahl, who holds the Daniel and Martina Lewis Chair. “This study has provided us with a concrete way of understanding the steps involved in mammary development and reveals a complexity that was not evident by most other methods. We are excited to share this with the broader research community.”

The authors plan to add data from more developmental time points to further develop the database of how cancer develops, with a goal of informing the development of more effective therapies.

 

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