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.

Breast-Specific Molecular Clocks Comprised of ELF5 Expression and Promoter Methylation Identify Individuals Susceptible to Cancer Initiation.

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.

Early growth response 2 (EGR2) is a novel regulator of the senescence programme.

Senescence, a state of stable growth arrest, plays an important role in ageing and age-related diseases in vivo. Although the INK4/ARF locus is known to be essential for senescence programmes, the key regulators driving p16 and ARF transcription remain largely underexplored. Using siRNA screening for modulators of the p16/pRB and ARF/p53/p21 pathways in deeply senescent human mammary epithelial cells (DS HMECs) and fibroblasts (DS HMFs), we identified EGR2 as a novel regulator of senescence. EGR2 expression is up-regulated during senescence, and its ablation by siRNA in DS HMECs and HMFs transiently reverses the senescent phenotype. We demonstrate that EGR2 activates the ARF and p16 promoters and directly binds to both the ARF and p16 promoters. Loss of EGR2 down-regulates p16 levels and increases the pool of p16- p21- 'reversed' cells in the population. Moreover, EGR2 overexpression is sufficient to induce senescence. Our data suggest that EGR2 is a direct transcriptional activator of the p16/pRB and ARF/p53/p21 pathways in senescence and a novel marker of senescence.

Evidence for accelerated aging in mammary epithelia of women carrying germline BRCA1 or BRCA2 mutations.

During aging in the human mammary gland, luminal epithelial cells lose lineage fidelity by expressing markers normally expressed in myoepithelial cells. We hypothesize that loss of lineage fidelity is a general manifestation of epithelia that are susceptible to cancer initiation. In the present study, we show that histologically normal breast tissue from younger women who are susceptible to breast cancer, as a result of harboring a germline mutation in BRCA1, BRCA2 or PALB2 genes, exhibits hallmarks of accelerated aging. These include proportionately increased luminal epithelial cells that acquired myoepithelial markers, decreased proportions of myoepithelial cells and a basal differentiation bias or failure of differentiation of cKit+ progenitors. High-risk luminal and myoepithelial cells are transcriptionally enriched for genes of the opposite lineage, inflammatory- and cancer-related pathways. We have identified breast-aging hallmarks that reflect a convergent biology of cancer susceptibility, regardless of the specific underlying genetic or age-dependent risk or the associated breast cancer subtype.

AXL Is a Driver of Stemness in Normal Mammary Gland and Breast Cancer.

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.

Breast Tissue Biology Expands the Possibilities for Prevention of Age-Related Breast Cancers.

Preventing breast cancer before it is able to form is an ideal way to stop breast cancer. However, there are limited existing options for prevention of breast cancer. Changes in the breast tissue resulting from the aging process contribute to breast cancer susceptibility and progression and may therefore provide promising targets for prevention. Here, we describe new potential targets, immortalization and inflammaging, that may be useful for prevention of age-related breast cancers. We also summarize existing studies of warfarin and metformin, current drugs used for non-cancerous diseases, that also may be repurposed for breast cancer prevention.

Superresolution microscopy reveals linkages between ribosomal DNA on heterologous chromosomes.

The spatial organization of the genome is enigmatic. Direct evidence of physical contacts between chromosomes and their visualization at nanoscale resolution has been limited. We used superresolution microscopy to demonstrate that ribosomal DNA (rDNA) can form linkages between chromosomes. We observed rDNA linkages in many different human cell types and demonstrated their resolution in anaphase. rDNA linkages are coated by the transcription factor UBF and their formation depends on UBF, indicating that they regularly occur between transcriptionally active loci. Overexpression of c-Myc increases rDNA transcription and the frequency of rDNA linkages, further suggesting that their formation depends on active transcription. Linkages persist in the absence of cohesion, but inhibition of topoisomerase II prevents their resolution in anaphase. We propose that linkages are topological intertwines occurring between transcriptionally active rDNA loci spatially colocated in the same nucleolar compartment. Our findings suggest that active DNA loci engage in physical interchromosomal connections that are an integral and pervasive feature of genome organization.

Experimental and pan-cancer genome analyses reveal widespread contribution of acrylamide exposure to carcinogenesis in humans.

Humans are frequently exposed to acrylamide, a probable human carcinogen found in commonplace sources such as most heated starchy foods or tobacco smoke. Prior evidence has shown that acrylamide causes cancer in rodents, yet epidemiological studies conducted to date are limited and, thus far, have yielded inconclusive data on association of human cancers with acrylamide exposure. In this study, we experimentally identify a novel and unique mutational signature imprinted by acrylamide through the effects of its reactive metabolite glycidamide. We next show that the glycidamide mutational signature is found in a full one-third of approximately 1600 tumor genomes corresponding to 19 human tumor types from 14 organs. The highest enrichment of the glycidamide signature was observed in the cancers of the lung (88% of the interrogated tumors), liver (73%), kidney (>70%), bile duct (57%), cervix (50%), and, to a lesser extent, additional cancer types. Overall, our study reveals an unexpectedly extensive contribution of acrylamide-associated mutagenesis to human cancers.

Delayed γH2AX foci disappearance in mammary epithelial cells from aged women reveals an age-associated DNA repair defect.

Aging is a degenerative process in which genome instability plays a crucial role. To gain insight into the link between organismal aging and DNA repair capacity, we analyzed DNA double-strand break (DSB) resolution efficiency in human mammary epithelial cells from 12 healthy donors of young and old ages. The frequency of DSBs was measured by quantifying the number of γH2AX foci before and after 1Gy of γ-rays and it was higher in cells from aged donors (ADs) at all times analyzed. At 24 hours after irradiation, ADs retained a significantly higher frequency of residual DSBs than young donors (YDs), which had already reached values close to basal levels. The kinetics of DSB induction and disappearance showed that cells from ADs and YDs repair DSBs with similar speed, although analysis of early times after irradiation indicate that a repair defect may lie within the firing of the DNA repair machinery in AD cells. Indeed, using a mathematical model we calculated a constant factor of delay affecting aged human epithelial cells repair kinetics. This defect manifests with the accumulation of DSBs that might eventually undergo illegitimate repair, thus posing a relevant threat to the maintenance of genome integrity in older individuals.

Genetic variation and radiation quality impact cancer promoting cellular phenotypes in response to HZE exposure.

There exists a wide degree of genetic variation within the normal human population which includes disease free individuals with heterozygote defects in major DNA repair genes. A lack of understanding of how this genetic variation impacts cellular phenotypes that inform cancer risk post heavy ion exposure poses a major limitation in developing personalized cancer risk assessment astronauts. We initiated a pilot study with Human Mammary Epithelial Cell strains (HMEC) derived from wild type, a p16 silenced derivative of wild type, and various genetic variants that were heterozygote for DNA repair genes; BRCA1, BRCA2 and ATM. Cells strains were exposed to different high and low LET radiation qualities to generate both simple and complex lesions and centrosome aberrations were examined as a surrogate marker of genomic instability and cancer susceptibility post different exposures. Our results indicate that centrosome aberration frequency is higher in the genetic variants under study. The aberration frequency increases with dose, complexity of the lesion generated by different radiation qualities and age of the individual. This increase in genomic instability correlates with elevated check-point activation post radiation exposure. These studies suggest that the influence of individual genetics on cell cycle regulation could modify the degree of early genomic instability in response to complex lesions and potentially define cancer predisposition in response to HZE exposure. These results will have significant implications in estimating cancer susceptibility in genetically variant individuals exposed to HZE particles.

Different culture media modulate growth, heterogeneity, and senescence in human mammary epithelial cell cultures.

The ability to culture normal human mammary epithelial cells (HMEC) greatly facilitates experiments that seek to understand both normal mammary cell biology and the many differences between normal and abnormal human mammary epithelia. To maximize in vivo relevance, the primary cell culture conditions should maintain cells in states that resemble in vivo as much as possible. Towards this goal, we compared the properties of HMEC strains from two different reduction mammoplasty tissues that were grown in parallel using different media and culture conditions. Epithelial organoids were initiated into three different media: two commonly used serum-free-media, MCDB 170-type (e.g. MEGM) and WIT-P, and a low stress media, M87A. Growth, lineage heterogeneity, p16 protein expression, and population doublings to senescence were measured for each culture condition. MCDB 170 caused rapid senescence and loss of heterogeneity within 2 to 3 passages, but some cultures went through the 1 to 2 month process of selection to generate clonal finite post-selection post-stasis cells. WIT-P caused impressive expansion of luminal cells in 2nd passage followed by their near complete disappearance by passage 4 and senescence shortly thereafter. M87A supported as much as twice the number of population doublings compared to either serum-free medium, and luminal and myoepithelial cells were present for as many as 8 passages. Thus, of the three media compared, WIT-P and MCDB 170 imposed rapid senescence and loss of lineage heterogeneity, phenotypes consistent with cells maintained in high-stress conditions, while M87A supported cultures that maintained multiple lineages and robust growth for up to 60 population doublings. In conjunction with previous studies examining the molecular properties of cultures grown in these media, we conclude that M87A medium is most able to support long-term culture of multiple lineages similar to in vivo conditions, thereby facilitating investigations of normal HMEC biology relevant to the mammary gland in situ.

Microenvironment-Induced Non-sporadic Expression of the AXL and cKIT Receptors Are Related to Epithelial Plasticity and Drug Resistance.

The existence of rare cancer cells that sporadically acquire drug-tolerance through epigenetic mechanisms is proposed as one mechanism that drives cancer therapy failure. Here we provide evidence that specific microenvironments impose non-sporadic expression of proteins related to epithelial plasticity and drug resistance. Microarrays of robotically printed combinatorial microenvironments of known composition were used to make cell-based functional associations between microenvironments, which were design-inspired by normal and tumor-burdened breast tissues, and cell phenotypes. We hypothesized that specific combinations of microenvironment constituents non-sporadically impose the induction of the AXL and cKIT receptor tyrosine kinase proteins, which are known to be involved in epithelial plasticity and drug-tolerance, in an isogenic human mammary epithelial cell (HMEC) malignant progression series. Dimension reduction analysis reveals type I collagen as a dominant feature, inducing expression of both markers in pre-stasis finite lifespan HMECs, and transformed non-malignant and malignant immortal cell lines. Basement membrane-associated matrix proteins, laminin-111 and type IV collagen, suppress AXL and cKIT expression in pre-stasis and non-malignant cells. However, AXL and cKIT are not suppressed by laminin-111 in malignant cells. General linear models identified key factors, osteopontin, IL-8, and type VIα3 collagen, which significantly upregulated AXL and cKIT, as well as a plasticity-related gene expression program that is often observed in stem cells and in epithelial-to-mesenchymal-transition. These factors are co-located with AXL-expressing cells in situ in normal and breast cancer tissues, and associated with resistance to paclitaxel. A greater diversity of microenvironments induced AXL and cKIT expression consistent with plasticity and drug-tolerant phenotypes in tumorigenic cells compared to normal or immortal cells, suggesting a reduced perception of microenvironment specificity in malignant cells. Microenvironment-imposed reprogramming could explain why resistant cells are seemingly persistent and rapidly adaptable to multiple classes of drugs. These results support the notion that specific microenvironments drive drug-tolerant cellular phenotypes and suggest a novel interventional avenue for preventing acquired therapy resistance.

High-Dimensional Phenotyping Identifies Age-Emergent Cells in Human Mammary Epithelia.

Aging is associated with tissue-level changes in cellular composition that are correlated with increased susceptibility to disease. Aging human mammary tissue shows skewed progenitor cell potency, resulting in diminished tumor-suppressive cell types and the accumulation of defective epithelial progenitors. Quantitative characterization of these age-emergent human cell subpopulations is lacking, impeding our understanding of the relationship between age and cancer susceptibility. We conducted single-cell resolution proteomic phenotyping of healthy breast epithelia from 57 women, aged 16-91 years, using mass cytometry. Remarkable heterogeneity was quantified within the two mammary epithelial lineages. Population partitioning identified a subset of aberrant basal-like luminal cells that accumulate with age and originate from age-altered progenitors. Quantification of age-emergent phenotypes enabled robust classification of breast tissues by age in healthy women. This high-resolution mapping highlighted specific epithelial subpopulations that change with age in a manner consistent with increased susceptibility to breast cancer.

Age-related gene expression in luminal epithelial cells is driven by a microenvironment made from myoepithelial cells.

Luminal epithelial cells in the breast gradually alter gene and protein expression with age, appearing to lose lineage-specificity by acquiring myoepithelial-like characteristics. We hypothesize that the luminal lineage is particularly sensitive to microenvironment changes, and age-related microenvironment changes cause altered luminal cell phenotypes. To evaluate the effects of different microenvironments on the fidelity of epigenetically regulated luminal and myoepithelial gene expression, we generated a set of lineage-specific probes for genes that are controlled through DNA methylation. Culturing primary luminal cells under conditions that favor myoepithelial propogation led to their reprogramming at the level of gene methylation, and to a more myoepithelial-like expression profile. Primary luminal cells' lineage-specific gene expression could be maintained when they were cultured as bilayers with primary myoepithelial cells. Isogenic stromal fibroblast co-cultures were unable to maintain the luminal phenotype. Mixed-age luminal-myoepithelial bilayers revealed that luminal cells adopt transcription and methylation patterns consistent with the chronological age of the myoepithelial cells. We provide evidence that the luminal epithelial phenotype is exquisitely sensitive to microenvironment conditions, and that states of aging are cell non-autonomously communicated through microenvironment cues over at least one cell diameter.

A lincRNA connected to cell mortality and epigenetically-silenced in most common human cancers.

Immortality is an essential characteristic of human carcinoma cells. We recently developed an efficient, reproducible method that immortalizes human mammary epithelial cells (HMEC) in the absence of gross genomic changes by targeting 2 critical senescence barriers. Consistent transcriptomic changes associated with immortality were identified using microarray analysis of isogenic normal finite pre-stasis, abnormal finite post-stasis, and immortal HMECs from 4 individuals. A total of 277 genes consistently changed in cells that transitioned from post-stasis to immortal. Gene ontology analysis of affected genes revealed biological processes significantly altered in the immortalization process. These immortalization-associated changes showed striking similarity to the gene expression changes seen in The Cancer Genome Atlas (TCGA) clinical breast cancer data. The most dramatic change in gene expression seen during the immortalization step was the downregulation of an unnamed, incompletely annotated transcript that we called MORT, for mortality, since its expression was closely associated with the mortal, finite lifespan phenotype. We show here that MORT (ZNF667-AS1) is expressed in all normal finite lifespan human cells examined to date and is lost in immortalized HMEC. MORT gene silencing at the mortal/immortal boundary was due to DNA hypermethylation of its CpG island promoter. This epigenetic silencing is also seen in human breast cancer cell lines and in a majority of human breast tumor tissues. The functional importance of DNA hypermethylation in MORT gene silencing is supported by the ability of 5-aza-2'-deoxycytidine to reactivate MORT expression. Analysis of TCGA data revealed deregulation of MORT expression due to DNA hypermethylation in 15 out of the 17 most common human cancers. The epigenetic silencing of MORT in a large majority of the common human cancers suggests a potential fundamental role in cellular immortalization during human carcinogenesis.

The senescent methylome and its relationship with cancer, ageing and germline genetic variation in humans.

BACKGROUND: Cellular senescence is a stable arrest of proliferation and is considered a key component of processes associated with carcinogenesis and other ageing-related phenotypes. Here, we perform methylome analysis of actively dividing and deeply senescent normal human epithelial cells. RESULTS: We identify senescence-associated differentially methylated positions (senDMPs) from multiple experiments using cells from one donor. We find that human senDMP epigenetic signatures are positively and significantly correlated with both cancer and ageing-associated methylation dynamics. We also identify germline genetic variants, including those associated with the p16INK4A locus, which are associated with the presence of in vivo senDMP signatures. Importantly, we also demonstrate that a single senDMP signature can be effectively reversed in a newly-developed protocol of transient senescence reversal. CONCLUSIONS: The senDMP signature has significant potential for understanding some of the key (epi)genetic etiological factors that may lead to cancer and age-related diseases in humans.

Delineating transcriptional networks of prognostic gene signatures refines treatment recommendations for lymph node-negative breast cancer patients.

The majority of women diagnosed with lymph node-negative breast cancer are unnecessarily treated with damaging chemotherapeutics after surgical resection. This highlights the importance of understanding and more accurately predicting patient prognosis. In the present study, we define the transcriptional networks regulating well-established prognostic gene expression signatures. We find that the same set of transcriptional regulators consistently lie upstream of both 'prognosis' and 'proliferation' gene signatures, suggesting that a central transcriptional network underpins a shared phenotype within these signatures. Strikingly, the master transcriptional regulators within this network predict recurrence risk for lymph node-negative breast cancer better than currently used multigene prognostic assays, particularly in estrogen receptor-positive patients. Simultaneous examination of p16(INK4A) expression, which predicts tumours that have bypassed cellular senescence, revealed that intermediate levels of p16(INK4A) correlate with an intact pRB pathway and improved survival. A combination of these master transcriptional regulators and p16(INK4A), termed the OncoMasTR score, stratifies tumours based on their proliferative and senescence capacity, facilitating a clearer delineation of lymph node-negative breast cancer patients at high risk of recurrence, and thus requiring chemotherapy. Furthermore, OncoMasTR accurately classifies over 60% of patients as 'low risk', an improvement on existing prognostic assays, which has the potential to reduce overtreatment in early-stage patients. Taken together, the present study provides new insights into the transcriptional regulation of cellular proliferation in breast cancer and provides an opportunity to enhance and streamline methods of predicting breast cancer prognosis.

Age and the means of bypassing stasis influence the intrinsic subtype of immortalized human mammary epithelial cells.

Based on molecular features, breast cancers are grouped into intrinsic subtypes that have different prognoses and therapeutic response profiles. With increasing age, breast cancer incidence increases, with hormone receptor-positive and other luminal-like subtype tumors comprising a majority of cases. It is not known at what stage of tumor progression subtype specification occurs, nor how the process of aging affects the intrinsic subtype. We examined subtype markers in immortalized human mammary epithelial cell lines established following exposure of primary cultured cell strains to a two-step immortalization protocol that targets the two main barriers to immortality: stasis (stress-associated senescence) and replicative senescence. Cell lines derived from epithelial cells obtained from non-tumorous pre- and post-menopausal breast surgery tissues were compared. Additionally, comparisons were made between lines generated using two different genetic interventions to bypass stasis: transduction of either an shRNA that down-regulated p16(INK4A), or overexpressed constitutive active cyclin D1/CDK2. In all cases, the replicative senescence barrier was bypassed by transduction of c-Myc. Cells from all resulting immortal lines exhibited normal karyotypes. Immunofluorescence, flow cytometry, and gene expression analyses of lineage-specific markers were used to categorize the intrinsic subtypes of the immortalized lines. Bypassing stasis with p16 shRNA in young strains generated cell lines that were invariably basal-like, but the lines examined from older strains exhibited some luminal features such as keratin 19 and estrogen receptor expression. Overexpression of cyclin D1/CDK2 resulted in keratin 19 positive, luminal-like cell lines from both young and old strains, and the lines examined from older strains exhibited estrogen receptor expression. Thus age and the method of bypassing stasis independently influence the subtype of immortalized human mammary epithelial cells.

Exome-wide mutation profile in benzo[a]pyrene-derived post-stasis and immortal human mammary epithelial cells.

Genetic mutations are known to drive cancer progression and certain tumors have mutation signatures that reflect exposures to environmental carcinogens. Benzo[a]pyrene (BaP) has a known mutation signature and has proven capable of inducing changes to DNA sequence that drives normal pre-stasis human mammary epithelial cells (HMEC) past a first tumor suppressor barrier (stasis) and toward immortality. We analyzed normal, pre-stasis HMEC, three independent BaP-derived post-stasis HMEC strains (184Aa, 184Be, 184Ce) and two of their immortal derivatives(184A1 and 184BE1) by whole exome sequencing. The independent post-stasis strains exhibited between 93 and 233 BaP-induced mutations in exons. Seventy percent of the mutations were C:G>A:T transversions, consistent with the known mutation spectrum of BaP. Mutations predicted to impact protein function occurred in several known and putative cancer drivers including p16, PLCG1, MED12, TAF1 in 184Aa; PIK3CG, HSP90AB1, WHSC1L1, LCP1 in 184Be and FANCA, LPP in 184Ce. Biological processes that typically harbor cancer driver mutations such as cell cycle, regulation of cell death and proliferation, RNA processing, chromatin modification and DNA repair were found to have mutations predicted to impact function in each of the post-stasis strains. Spontaneously immortalized HMEC lines derived from two of the BaP-derived post-stasis strains shared greater than 95% of their BaP-induced mutations with their precursor cells. These immortal HMEC had 10 or fewer additional point mutations relative to their post-stasis precursors, but acquired chromosomal anomalies during immortalization that arose independent of BaP. The results of this study indicate that acute exposures of HMEC to high dose BaP recapitulate mutation patterns of human tumors and can induce mutations in a number of cancer driver genes.