The receptor tyrosine kinase AXL is associated with epithelial plasticity in several solid tumors including breast cancer and AXL-targeting agents are currently in clinical trials. We hypothesized that AXL is a driver of stemness traits in cancer by co-option of a regulatory function normally reserved for stem cells. AXL-expressing cells in human mammary epithelial ducts co-expressed markers associated with multipotency, and AXL inhibition abolished colony formation and self-maintenance activities while promoting terminal differentiation in vitro. Axl-null mice did not exhibit a strong developmental phenotype, but enrichment of Axl+ cells was required for mouse mammary gland reconstitution upon transplantation, and Axl-null mice had reduced incidence of Wnt1-driven mammary tumors. An AXL-dependent gene signature is a feature of transcriptomes in basal breast cancers and reduced patient survival irrespective of subtype. Our interpretation is that AXL regulates access to epithelial plasticity programs in MaSCs and, when co-opted, maintains acquired stemness in breast cancer cells.

A robust breast cancer prevention strategy requires risk assessment biomarkers for early detection. We show that expression of ELF5, a transcription factor critical for normal mammary development, is downregulated in mammary luminal epithelia with age. DNA methylation of the ELF5 promoter is negatively correlated with expression in an age-dependent manner. Both ELF5 methylation and gene expression were used to build biological clocks to estimate chronological ages of mammary epithelia. ELF5 clock-based estimates of biological age in luminal epithelia from average-risk women were within three years of chronological age. Biological ages of breast epithelia from BRCA1 or BRCA2 mutation carriers, who were high risk for developing breast cancer, suggested they were accelerated by two decades relative to chronological age. The ELF5 DNA methylation clock had better performance at predicting biological age in luminal epithelial cells as compared with two other epigenetic clocks based on whole tissues. We propose that the changes in ELF5 expression or ELF5-proximal DNA methylation in luminal epithelia are emergent properties of at-risk breast tissue and constitute breast-specific biological clocks.Prevention Relevance: ELF5 expression or DNA methylation level at the ELF5 promoter region can be used as breast-specific biological clocks to identify women at higher than average risk of breast cancer.

Effects from aging in single cells are heterogenous, whereas at the organ- and tissue-levels aging phenotypes tend to appear as stereotypical changes. The mammary epithelium is a bilayer of two major phenotypically and functionally distinct cell lineages: luminal epithelial and myoepithelial cells. Mammary luminal epithelia exhibit substantial stereotypical changes with age that merit attention because these cells are the putative cells-of-origin for breast cancers. We hypothesize that effects from aging that impinge upon maintenance of lineage fidelity increase susceptibility to cancer initiation. We generated and analyzed transcriptomes from primary luminal epithelial and myoepithelial cells from younger 55 y women. In addition to age-dependent directional changes in gene expression, we observed increased transcriptional variance with age that contributed to genome-wide loss of lineage fidelity. Age-dependent variant responses were common to both lineages, whereas directional changes were almost exclusively detected in luminal epithelia and involved altered regulation of chromatin and genome organizers such as SATB1. Epithelial expression variance of gap junction protein GJB6 increased with age, and modulation of GJB6 expression in heterochronous co-cultures revealed that it provided a communication conduit from myoepithelial cells that drove directional change in luminal cells. Age-dependent luminal transcriptomes comprised a prominent signal that could be detected in bulk tissue during aging and transition into cancers. A machine learning classifier based on luminal-specific aging distinguished normal from cancer tissue and was highly predictive of breast cancer subtype. We speculate that luminal epithelia are the ultimate site of integration of the variant responses to aging in their surrounding tissue, and that their emergent phenotype both endows cells with the ability to become cancer-cells-of-origin and represents a biosensor that presages cancer susceptibility.

Age is the major risk factor in most carcinomas, yet little is known about how proteomes change with age in any human epithelium. We present comprehensive proteomes comprised of >9,000 total proteins and >15,000 phosphopeptides from normal primary human mammary epithelia at lineage resolution from ten women ranging in age from 19 to 68 years. Data were quality controlled and results were biologically validated with cell-based assays. Age-dependent protein signatures were identified using differential expression analyses and weighted protein co-expression network analyses. Upregulation of basal markers in luminal cells, including KRT14 and AXL, were a prominent consequence of aging. PEAK1 was identified as an age-dependent signaling kinase in luminal cells, which revealed a potential age-dependent vulnerability for targeted ablation. Correlation analyses between transcriptome and proteome revealed age-associated loss of proteostasis regulation. Age-dependent proteome changes in the breast epithelium identified heretofore unknown potential therapeutic targets for reducing breast cancer susceptibility.

Breast cancer is predominantly an age-related disease, with aging serving as the most significant risk factor, compounded by germline mutations in high-risk genes like BRCA1/2. Aging induces architectural changes in breast tissue, particularly affecting luminal epithelial cells by diminishing lineage-specific molecular profiles and adopting myoepithelial-like characteristics. ELF5 is an important transcription factor for both normal breast and breast cancer development. This review focuses on the role of ELF5 in normal breast development, its altered expression throughout aging, and its implications in cancer. It discusses the lineage-specific expression of ELF5, its regulatory mechanisms, and its potential as a biomarker for breast-specific biological age and cancer risk.

Summary: Aging causes molecular changes that manifest as stereotypical phenotypes yet aging-associated diseases progress only in certain individuals. At lineage-specific resolution, we show how stereotyped and variant responses are integrated in mammary epithelia. Age-dependent directional changes in gene expression and DNA methylation (DNAm) occurred almost exclusively in luminal cells and implicated genome organizers SATB1 and CTCF . DNAm changes were robust indicators of aging luminal cells, and were either directly (anti-)correlated with expression changes or served as priming events for subsequent dysregulation, such as demethylation of ESR1 -binding regions in DNAm-regulatory CXXC5 in older luminal cells and luminal-subtype cancers. Variance-driven changes in the transcriptome of both luminal and myoepithelial lineages further contributed to age-dependent loss of lineage fidelity. The pathways affected by transcriptomic and DNAm changes during aging are commonly linked with breast cancer, and together with the differential variability found across individuals, influence aging-associated cancer susceptibility in a subtype-specific manner.