Loss of epigenetic suppression of retrotransposons with oncogenic potential in aging mammary luminal epithelial cells.

A primary function of DNA methylation in mammalian genomes is to repress transposable elements (TEs). The widespread methylation loss that is commonly observed in cancer cells results in the loss of epigenetic repression of TEs. The aging process is similarly characterized by changes to the methylome. However, the impact of these epigenomic alterations on TE silencing and the functional consequences of this have remained unclear. To assess the epigenetic regulation of TEs in aging, we profiled DNA methylation in human mammary luminal epithelial cells (LEps)-a key cell lineage implicated in age-related breast cancers-from younger and older women. We report here that several TE subfamilies function as regulatory elements in normal LEps, and a subset of these display consistent methylation changes with age. Methylation changes at these TEs occurred at lineage-specific transcription factor binding sites, consistent with loss of lineage specificity. Whereas TEs mainly showed methylation loss, CpG islands (CGIs) that are targets of the Polycomb repressive complex 2 (PRC2) show a gain of methylation in aging cells. Many TEs with methylation loss in aging LEps have evidence of regulatory activity in breast cancer samples. We furthermore show that methylation changes at TEs impact the regulation of genes associated with luminal breast cancers. These results indicate that aging leads to DNA methylation changes at TEs that undermine the maintenance of lineage specificity, potentially increasing susceptibility to breast cancer.

Sustained postconfluent culture of human mammary epithelial cells enriches for luminal and c-Kit+ subtypes.

BACKGROUND: A challenge in human mammary epithelial cell (HMEC) culture is sustaining the representation of competing luminal, myoepithelial, and progenitor lineages over time. As cells replicate in culture, myoepithelial cells come to dominate the composition of the culture with serial passaging. This drift in composition presents a challenge for studying luminal and progenitor cells, which are prospective cells of origin for most breast cancer subtypes. METHODS: We demonstrate the use of postconfluent culture on HMECs. Postconfluent culture entails culturing HMECs for 2-5 weeks without passaging but maintaining frequent feedings in low-stress M87A culture medium. In contrast, standard HMEC culture entails enzymatic subculturing every 3-5 days to maintain subconfluent density. RESULTS: When compared to standard HMEC culture, postconfluent culture yields increased proportions of luminal cells and c-Kit+ progenitor cells. Postconfluent cultures develop a distinct multilayered morphology with individual cells showing decreased physical deformability as compared to cells in standard culture. Gene expression analysis of postconfluent cells shows increased expression of lineage-specific markers and extracellular matrix components. CONCLUSIONS: Postconfluent culture is a novel, useful strategy for altering the lineage composition of HMECs, by increasing the proportional representation of luminal and progenitor cells. We speculate that postconfluent culture creates a microenvironment with cellular composition closer to the physiological state and eases the isolation of scarce cell subtypes. As such, postconfluent culture is a valuable tool for researchers using HMECs for breast cancer research.

Cellular and molecular mechanisms of breast cancer susceptibility.

There is a plethora of recognized risk factors for breast cancer (BC) with poorly understood or speculative biological mechanisms. The lack of prevention options highlights the importance of understanding the mechanistic basis of cancer susceptibility and finding new targets for breast cancer prevention. Until now, we have understood risk and cancer susceptibility primarily through the application of epidemiology and assessing outcomes in large human cohorts. Relative risks are assigned to various human behaviors and conditions, but in general the associations are weak and there is little understanding of mechanism. Aging is by far the greatest risk factor for BC, and there are specific forms of inherited genetic risk that are well-understood to cause BC. We propose that bringing focus to the biology underlying these forms of risk will illuminate biological mechanisms of BC susceptibility.

Adaptation to Hypoxia May Promote Therapeutic Resistance to Androgen Receptor Inhibition in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) surpasses other BC subtypes as the most challenging to treat due to its lack of traditional BC biomarkers. Nearly 30% of TNBC patients express the androgen receptor (AR), and the blockade of androgen production and AR signaling have been the cornerstones of therapies for AR-positive TNBC. However, the majority of women are resistant to AR-targeted therapy, which is a major impediment to improving outcomes for the AR-positive TNBC subpopulation. The hypoxia signaling cascade is frequently activated in the tumor microenvironment in response to low oxygen levels; activation of the hypoxia signaling cascade allows tumors to survive despite hypoxia-mediated interference with cellular metabolism. The activation of hypoxia signaling networks in TNBC promotes resistance to most anticancer drugs including AR inhibitors. The activation of hypoxia network signaling occurs more frequently in TNBC compared to other BC subtypes. Herein, we examine the (1) interplay between hypoxia signaling networks and AR and (2) whether hypoxia and hypoxic stress adaptive pathways promote the emergence of resistance to therapies that target AR. We also pose the well-supported question, "Can the efficacy of androgen-/AR-targeted treatments be enhanced by co-targeting hypoxia?" By critically examining the evidence and the complex entwinement of these two oncogenic pathways, we argue that the simultaneous targeting of androgen biosynthesis/AR signaling and hypoxia may enhance the sensitivity of AR-positive TNBCs to AR-targeted treatments, derail the emergence of therapy resistance, and improve patient outcomes.

Changes in Immune Cell Types with Age in Breast are Consistent with a Decline in Immune Surveillance and Increased Immunosuppression.

A majority of breast cancers (BC) are age-related and we seek to determine what cellular and molecular changes occur in breast tissue with age that make women more susceptible to cancer initiation. Immune-epithelial cell interactions are important during mammary gland development and the immune system plays an important role in BC progression. The composition of human immune cell populations is known to change in peripheral blood with age and in breast tissue during BC progression. Less is known about changes in immune populations in normal breast tissue and how their interactions with mammary epithelia change with age. We quantified densities of T cells, B cells, and macrophage subsets in pathologically normal breast tissue from 122 different women who ranged in age from 24 to 74 years old. Donor-matched peripheral blood from a subset of 20 donors was analyzed by flow cytometry. Tissue immune cell densities and localizations relative to the epithelium were quantified in situ with machine learning-based image analyses of multiplex immunohistochemistry-stained tissue sections. In situ results were corroborated with flow cytometry analyses of peri-epithelial immune cells from primary breast tissue preparations and transcriptome analyses of public data from bulk tissue reduction mammoplasties. Proportions of immune cell subsets in breast tissue and donor-matched peripheral blood were not correlated. Density (cells/mm2) of T and B lymphocytes in situ decreased with age. T cells and macrophages preferentially localized near or within epithelial bilayers, rather than the intralobular stroma. M2 macrophage density was higher than M1 macrophage density and this difference was due to higher density of M2 in the intralobular stroma. Transcriptional signature analyses suggested age-dependent decline in adaptive immune cell populations and functions and increased innate immune cell activity. T cells and macrophages are so intimately associated with the epithelia that they are embedded within the bilayer, suggesting an important role for immune-epithelial cell interactions. Age-associated decreased T cell density in peri-epithelial regions, and increased M2 macrophage density in intralobular stroma suggests the emergence of a tissue microenvironment that is simultaneously immune-senescent and immunosuppressive with age.

Programmed synthesis of three-dimensional tissues.

Reconstituting tissues from their cellular building blocks facilitates the modeling of morphogenesis, homeostasis and disease in vitro. Here we describe DNA-programmed assembly of cells (DPAC), a method to reconstitute the multicellular organization of organoid-like tissues having programmed size, shape, composition and spatial heterogeneity. DPAC uses dissociated cells that are chemically functionalized with degradable oligonucleotide 'Velcro', allowing rapid, specific and reversible cell adhesion to other surfaces coated with complementary DNA sequences. DNA-patterned substrates function as removable and adhesive templates, and layer-by-layer DNA-programmed assembly builds arrays of tissues into the third dimension above the template. DNase releases completed arrays of organoid-like microtissues from the template concomitant with full embedding in a variety of extracellular matrix (ECM) gels. DPAC positions subpopulations of cells with single-cell spatial resolution and generates cultures several centimeters long. We used DPAC to explore the impact of ECM composition, heterotypic cell-cell interactions and patterns of signaling heterogeneity on collective cell behaviors.

A strategy for tissue self-organization that is robust to cellular heterogeneity and plasticity.

Developing tissues contain motile populations of cells that can self-organize into spatially ordered tissues based on differences in their interfacial surface energies. However, it is unclear how self-organization by this mechanism remains robust when interfacial energies become heterogeneous in either time or space. The ducts and acini of the human mammary gland are prototypical heterogeneous and dynamic tissues comprising two concentrically arranged cell types. To investigate the consequences of cellular heterogeneity and plasticity on cell positioning in the mammary gland, we reconstituted its self-organization from aggregates of primary cells in vitro. We find that self-organization is dominated by the interfacial energy of the tissue-ECM boundary, rather than by differential homo- and heterotypic energies of cell-cell interaction. Surprisingly, interactions with the tissue-ECM boundary are binary, in that only one cell type interacts appreciably with the boundary. Using mathematical modeling and cell-type-specific knockdown of key regulators of cell-cell cohesion, we show that this strategy of self-organization is robust to severe perturbations affecting cell-cell contact formation. We also find that this mechanism of self-organization is conserved in the human prostate. Therefore, a binary interfacial interaction with the tissue boundary provides a flexible and generalizable strategy for forming and maintaining the structure of two-component tissues that exhibit abundant heterogeneity and plasticity. Our model also predicts that mutations affecting binary cell-ECM interactions are catastrophic and could contribute to loss of tissue architecture in diseases such as breast cancer.

Breast Cancer beyond the Age of Mutation.

Age is the greatest risk factor for breast cancer, but the reasons underlying this association are unclear. While there is undeniably a genetic component to all cancers, the accumulation of mutations with age is insufficient to explain the age-dependent increase in breast cancer incidence. In this viewpoint, we propose a multilevel framework to better understand the respective roles played by somatic mutation, microenvironment, and epigenetics making women more susceptible to breast cancer with age. The process of aging is associated with gradual breast tissue changes that not only corrupt the tumor-suppressive activity of normal tissue but also impose age-specific epigenetic changes that alter gene expression, thus reinforcing cellular phenotypes that are associated with a continuum of age-related tissue microenvironments. The evidence discussed here suggests that while the riddle of whether epigenetics drives microenvironmental changes, or whether changes in the microenvironment alter heritable cellular memory has not been solved, a path has been cleared enabling functional analysis leading to the prediction of key nodes in the network that link the microenvironment with the epigenome. The hypothesis that the accumulation of somatic mutations with age drives the age-related increase in breast cancer incidence, if correct, has a somewhat nihilistic conclusion, namely that cancers will be impossible to avoid. Alternatively, if microenvironment-driven epigenetic changes are the key to explaining susceptibility to age-related breast cancers, then there is hope that primary prevention is possible because epigenomes are relatively malleable.

Self-organization is a dynamic and lineage-intrinsic property of mammary epithelial cells.

Loss of organization is a principle feature of cancers; therefore it is important to understand how normal adult multilineage tissues, such as bilayered secretory epithelia, establish and maintain their architectures. The self-organization process that drives heterogeneous mixtures of cells to form organized tissues is well studied in embryology and with mammalian cell lines that were abnormal or engineered. Here we used a micropatterning approach that confined cells to a cylindrical geometry combined with an algorithm to quantify changes of cellular distribution over time to measure the ability of different cell types to self-organize relative to each other. Using normal human mammary epithelial cells enriched into pools of the two principal lineages, luminal and myoepithelial cells, we demonstrated that bilayered organization in mammary epithelium was driven mainly by lineage-specific differential E-cadherin expression, but that P-cadherin contributed specifically to organization of the myoepithelial layer. Disruption of the actomyosin network or of adherens junction proteins resulted in either prevention of bilayer formation or loss of preformed bilayers, consistent with continual sampling of the local microenvironment by cadherins. Together these data show that self-organization is an innate and reversible property of communities of normal adult human mammary epithelial cells.

ELF5: A Molecular Clock for Breast Aging and 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.

Functional delineation of the luminal epithelial microenvironment in breast using cell-based screening in combinatorial microenvironments.

Microenvironment signals are potent determinants of cell fate and arbiters of tissue homeostasis, however understanding how different microenvironment factors coordinately regulate cellular phenotype has been experimentally challenging. Here we used a high-throughput microenvironment microarray comprised of 2640 unique pairwise signals to identify factors that support proliferation and maintenance of primary human mammary luminal epithelial cells. Multiple microenvironment factors that modulated luminal cell number were identified, including: HGF, NRG1, BMP2, CXCL1, TGFB1, FGF2, PDGFB, RANKL, WNT3A, SPP1, HA, VTN, and OMD. All of these factors were previously shown to modulate luminal cell numbers in painstaking mouse genetics experiments, or were shown to have a role in breast cancer, demonstrating the relevance and power of our high-dimensional approach to dissect key microenvironmental signals. RNA-sequencing of primary epithelial and stromal cell lineages identified the cell types that express these signals and the cognate receptors in vivo. Cell-based functional studies confirmed which effects from microenvironment factors were reproducible and robust to individual variation. Hepatocyte growth factor (HGF) was the factor most robust to individual variation and drove expansion of luminal cells via cKit+ progenitor cells, which expressed abundant MET receptor. Luminal cells from women who are genetically high risk for breast cancer had significantly more MET receptor and may explain the characteristic expansion of the luminal lineage in those women. In ensemble, our approach provides proof of principle that microenvironment signals that control specific cellular states can be dissected with high-dimensional cell-based approaches.

A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens.

The plasma membrane is enriched for receptors and signaling proteins that are accessible from the extracellular space for pharmacological intervention. Here we conducted a series of CRISPR screens using human cell surface proteome and integrin family libraries in multiple cancer models. Our results identified ITGAV (integrin αV) and its heterodimer partner ITGB5 (integrin ß5) as the essential integrin α/ß pair for cancer cell expansion. High-density CRISPR gene tiling further pinpointed the integral pocket within the ß-propeller domain of ITGAV for integrin αVß5 dimerization. Combined with in silico compound docking, we developed a CRISPR-Tiling-Instructed Computer-Aided (CRISPR-TICA) pipeline for drug discovery and identified Cpd_AV2 as a lead inhibitor targeting the ß-propeller central pocket of ITGAV. Cpd_AV2 treatment led to rapid uncoupling of integrin αVß5 and cellular apoptosis, providing a unique class of therapeutic action that eliminates the integrin signaling via heterodimer dissociation. We also foresee the CRISPR-TICA approach to be an accessible method for future drug discovery studies.

Configurational entropy is an intrinsic driver of tissue structural heterogeneity.

Tissues comprise ordered arrangements of cells that can be surprisingly disordered in their details. How the properties of single cells and their microenvironment contribute to the balance between order and disorder at the tissue-scale remains poorly understood. Here, we address this question using the self-organization of human mammary organoids as a model. We find that organoids behave like a dynamic structural ensemble at the steady state. We apply a maximum entropy formalism to derive the ensemble distribution from three measurable parameters - the degeneracy of structural states, interfacial energy, and tissue activity (the energy associated with positional fluctuations). We link these parameters with the molecular and microenvironmental factors that control them to precisely engineer the ensemble across multiple conditions. Our analysis reveals that the entropy associated with structural degeneracy sets a theoretical limit to tissue order and provides new insight for tissue engineering, development, and our understanding of disease progression.

Comprehensive single cell aging atlas of mammary tissues reveals shared epigenomic and transcriptomic signatures of aging and cancer.

Aging is the greatest risk factor for breast cancer; however, how age-related cellular and molecular events impact cancer initiation is unknown. We investigate how aging rewires transcriptomic and epigenomic programs of mouse mammary glands at single cell resolution, yielding a comprehensive resource for aging and cancer biology. Aged epithelial cells exhibit epigenetic and transcriptional changes in metabolic, pro-inflammatory, or cancer-associated genes. Aged stromal cells downregulate fibroblast marker genes and upregulate markers of senescence and cancer-associated fibroblasts. Among immune cells, distinct T cell subsets (Gzmk+, memory CD4+, γδ) and M2-like macrophages expand with age. Spatial transcriptomics reveal co-localization of aged immune and epithelial cells in situ. Lastly, transcriptional signatures of aging mammary cells are found in human breast tumors, suggesting mechanistic links between aging and cancer. Together, these data uncover that epithelial, immune, and stromal cells shift in proportions and cell identity, potentially impacting cell plasticity, aged microenvironment, and neoplasia risk.

Preneoplastic stromal cells promote BRCA1-mediated breast tumorigenesis.

Women with germline BRCA1 mutations (BRCA1+/mut) have increased risk for hereditary breast cancer. Cancer initiation in BRCA1+/mut is associated with premalignant changes in breast epithelium; however, the role of the epithelium-associated stromal niche during BRCA1-driven tumor initiation remains unclear. Here we show that the premalignant stromal niche promotes epithelial proliferation and mutant BRCA1-driven tumorigenesis in trans. Using single-cell RNA sequencing analysis of human preneoplastic BRCA1+/mut and noncarrier breast tissues, we show distinct changes in epithelial homeostasis including increased proliferation and expansion of basal-luminal intermediate progenitor cells. Additionally, BRCA1+/mut stromal cells show increased expression of pro-proliferative paracrine signals. In particular, we identify pre-cancer-associated fibroblasts (pre-CAFs) that produce protumorigenic factors including matrix metalloproteinase 3 (MMP3), which promotes BRCA1-driven tumorigenesis in vivo. Together, our findings demonstrate that precancerous stroma in BRCA1+/mut may elevate breast cancer risk through the promotion of epithelial proliferation and an accumulation of luminal progenitor cells with altered differentiation.

Evidence for a stem cell hierarchy in the adult human breast.

Cellular pathways that contribute to adult human mammary gland architecture and lineages have not been previously described. In this study, we identify a candidate stem cell niche in ducts and zones containing progenitor cells in lobules. Putative stem cells residing in ducts were essentially quiescent, whereas the progenitor cells in the lobules were more likely to be actively dividing. Cells from ducts and lobules collected under the microscope were functionally characterized by colony formation on tissue culture plastic, mammosphere formation in suspension culture, and morphogenesis in laminin-rich extracellular matrix gels. Staining for the lineage markers keratins K14 and K19 further revealed multipotent cells in the stem cell zone and three lineage-restricted cell types outside this zone. Multiparameter cell sorting and functional characterization with reference to anatomical sites in situ confirmed this pattern. The proposal that the four cell types are indeed constituents of an as of yet undescribed stem cell hierarchy was assessed in long-term cultures in which senescence was bypassed. These findings identify an adult human breast ductal stem cell activity and its earliest descendants.

High-Throughput Microenvironment Microarray (MEMA) High-Resolution Imaging.

The interaction between cells and their surrounding microenvironment has a crucial role in determining cell fate. In many pathological conditions, the microenvironment drives disease progression as well as therapeutic resistance. A number of challenges arise for researchers examining these cell-microenvironment interactions: (1) Tissue microenvironments are combinatorial and dynamic systems, and in pathological situations like cancer, microenvironments become infamously chaotic and highly heterogeneous. (2) Cells exhibit heterogeneous phenotypes, and even rare cell subpopulations can have a substantial role in tissue homeostasis and disease progression. This chapter discusses technical aspects relevant to dissecting cell-microenvironment interaction using the Microenvironment Microarray (MEMA) platform, which is a cell-based functional high-throughput screening of interactions between cells and combinatorial microenvironments at the single-cell level. MEMA provides insights into how cell phenotype and function is elicited by microenvironmental components. In this chapter, we describe automating a high-throughput and high-resolution imaging pipeline for single-cell-resolution analysis.

Protocol for computationally evaluating the loss of stoichiometry and coordinated expression of proteins.

Dysregulation of the transcriptional or translational machinery can alter the stoichiometry of multiprotein complexes and occurs in natural processes such as aging. Loss of stoichiometry has been shown to alter protein complex functions. We provide a protocol and associated code that use omics data to quantify these stoichiometric changes via statistical dispersion utilizing the interquartile range of expression values per grouping variable. This descriptive statistical approach enables the quantification of stoichiometry changes without additional data acquisition. For complete details on the use and execution of this protocol, please refer to Hinz et al. (2021).

NCI's publication affiliation conundrum: Reframing innovation to incentivize an equitable path for advocate representation.

Advocacy engagement has been at the forefront of National Cancer Institute (NCI) efforts to advance scientific discoveries and transform medical interventions. Nonetheless, the journey for advocates has been uneven. Case in Point: NCI publication affiliation rules of engagement pose unique equity challenges while raising questions about structural representation in biomedical research. Abiding by the core rationale that publication affiliation should be tailored to employment status, the NCI has systematically denied research advocate volunteers the opportunity to specifically list NCI as an institutional affiliation on academic publications. Unpacking advocate NCI publication affiliation restrictions and its links with advocacy heritage preservation and convergent science goals poses unique diversity, equity, and inclusion challenges and opportunities. Improving the quality of structural representation in biomedical research requires new theories of action and flexible planning to advance, promote and build capacity for strategic advocacy inclusion and equity within publication affiliation initiatives. Here we highlight several opportunities for how leadership might formulate a radically different vision for NCI's approach. This perspective interrogates the best way forward for ensuring that biomedical employee and volunteer advocate workforce publication affiliation intersections are characterized by increased creativity and representation parity. Imbuing the scientist and clinical researcher archetype with social dimensions, we join NCI critical thinkers in urging employees, funded academics, and volunteer citizen scientists to collectively assume the role as paladins of science and integrity who view the triumphs of making a difference in science alongside the social responsibility of promoting transdisciplinary professionalism and the democratization of science.

The DARC Side of Inflamm-Aging: Duffy Antigen Receptor for Chemokines (DARC/ACKR1) as a Potential Biomarker of Aging, Immunosenescence, and Breast Oncogenesis among High-Risk Subpopulations.

The proclivity of certain pre-malignant and pre-invasive breast lesions to progress while others do not continues to perplex clinicians. Clinicians remain at a crossroads with effectively managing the high-risk patient subpopulation owing to the paucity of biomarkers that can adequately risk-stratify and inform clinical decisions that circumvent unnecessary administration of cytotoxic and invasive treatments. The immune system mounts the most important line of defense against tumorigenesis and progression. Unfortunately, this defense declines or "ages" over time-a phenomenon known as immunosenescence. This results in "inflamm-aging" or the excessive infiltration of pro-inflammatory chemokines, which alters the leukocyte composition of the tissue microenvironment, and concomitant immunoediting of these leukocytes to diminish their antitumor immune functions. Collectively, these effects can foster the sequelae of neoplastic transformation and progression. The erythrocyte cell antigen, Duffy antigen receptor for chemokines(DARC/ACKR1), binds and internalizes chemokines to maintain homeostatic levels and modulate leukocyte trafficking. A negative DARC status is highly prevalent among subpopulations of West African genetic ancestry, who are at higher risk of developing breast cancer and disease progression at a younger age. However, the role of DARC in accelerated inflamm-aging and malignant transformation remains underexplored. Herein, we review compelling evidence suggesting that DARC may be protective against inflamm-aging and, therefore, reduce the risk of a high-risk lesion progressing to malignancy. We also discuss evidence supporting that immunotherapeutic intervention-based on DARC status-among high-risk subpopulations may evade malignant transformation and progression. A closer look into this unique role of DARC could glean deeper insight into the immune response profile of individual high-risk patients and their predisposition to progress as well as guide the administration of more "cyto-friendly" immunotherapeutic intervention to potentially "turn back the clock" on inflamm-aging-mediated oncogenesis and progression.