Ethnicity-dependent and -independent heterogeneity in healthy normal breast hierarchy impacts tumor characterization

Recent reports of widespread genetic variation affecting regulation of gene expression raise the possibility of significant inter-individual differences in stem-progenitor-mature cell hierarchy in adult organs. This has not been explored because of paucity of methods to quantitatively assess subpopulation of normal epithelial cells on individual basis. We report the remarkable inter-individual differences in differentiation capabilities as documented by phenotypic heterogeneity in stem-progenitor-mature cell hierarchy of the normal breast. Ethnicity and genetic predisposition are partly responsible for this heterogeneity, evidenced by the finding that CD44+/CD24- and PROCR+/EpCAM- multi-potent stem cells were elevated significantly in African American women compared with Caucasians. ALDEFLUOR+ luminal stem/progenitor cells were lower in BRCA1-mutation carriers compared with cells from healthy donors (p = 0.0014). Moreover, tumor and adjoining-normal breast cells of the same patients showed distinct CD49f+/EpCAM+ progenitor, CD271+/EpCAM- basal, and ALDEFLUOR+ cell profiles. These inter-individual differences in the rate of differentiation in the normal breast may contribute to a substantial proportion of transcriptome, epigenome, and signaling pathway alterations and consequently has the potential to spuriously magnify the extent of documented tumor-specific gene expression. Therefore, comparative analysis of phenotypically defined subpopulations of normal and tumor cells on an individual basis may be required to identify cancer-specific aberrations

Cancer impacts microRNA expression, release and function in cardiac and skeletal muscle

Circulating microRNAs are emerging as important biomarkers of various diseases including cancer. Intriguingly, circulating levels of several microRNAs are lower in cancer patients compared with healthy individuals. In this study, we tested the hypothesis that a circulating microRNA might serve as a surrogate of the effects of cancer on microRNA expression or release in distant organs. Here we report that circulating levels of the muscle-enriched miR-486 is lower in breast cancer patients compared with healthy individuals, and that this difference is replicated faithfully in MMTV-PyMT and MMTV-Her2 transgenic mouse models of breast cancer. In tumor-bearing mice, levels of miR-486 were relatively reduced in muscle, where there was elevated expression of the miR-486 target genes PTEN and FOXO1A and dampened signaling through the PI3K/AKT pathway. Skeletal muscle expressed lower levels of the transcription factor MyoD which controls miR-486 expression. Conditioned media (CM) obtained from MMTV-PyMT and MMTV-Her2/Neu tumor cells cultured in vitro was sufficient to elicit reduced levels of miR-486 and increased PTEN and FOXO1A expression in C2C12 murine myoblasts. Cytokine analysis implicated TNFα and four additional cytokines as mediators of miR-486 expression in CM-treated cells. Since miR-486 is a potent modulator of PI3K/AKT signaling and the muscle-enriched transcription factor network in cardiac/skeletal muscle, our findings implicated TNFα-dependent miRNA circuitry in muscle differentiation and survival pathways in cancer

Distinct Effects of Adipose-derived Stem Cells and Adipocytes on Normal and Cancer Cell Hierarchy

Adipose-derived stem cells (ASC) have received considerable attention in oncology because of the known direct link between obesity and cancer as well as the use of ASCs in reconstructive surgery after tumor ablation. Previous studies have documented how cancer cells commandeer ASCs to support their survival by altering extracellular matrix composition and stiffness, migration, and metastasis. This study focused on delineating the effects of ASCs and adipocytes on the self-renewal of stem/progenitor cells and hierarchy of breast epithelial cells. The immortalized breast epithelial cell line MCF10A, ductal carcinoma in situ (DCIS) cell lines MCF10DCIS.com and SUM225, and MCF10A-overexpressing SRC oncogene were examined using a mammosphere assay and flow cytometry for the effects of ASCs on their self-renewal and stem-luminal progenitor-differentiated cell surface marker profiles. Interestingly, ASCs promoted the self-renewal of all cell types except SUM225. ASC coculture or treatment with ASC conditioned media altered the number of CD49fhigh/EpCAMlow basal/stem-like and CD49fmedium/EpCAMmedium luminal progenitor cells. Among multiple factors secreted by ASCs, IFNγ and hepatocyte growth factor (HGF) displayed unique actions on epithelial cell hierarchy. IFNγ increased stem/progenitor-like cells while simultaneously reducing the size of mammospheres, whereas HGF increased the size of mammospheres with an accompanying increase in luminal progenitor cells. ASCs expressed higher levels of HGF, whereas adipocytes expressed higher levels of IFNγ. As luminal progenitor cells are believed to be prone for transformation, IFNγ and HGF expression status of ASCs may influence susceptibility for developing breast cancer as well as on outcomes of autologous fat transplantation on residual/dormant tumor cells. Implications: This study suggests that the ratio of ASCs to adipocytes influences cancer cell hierarchy, which may impact incidence and progression

Normal Breast-Derived Epithelial Cells with Luminal and Intrinsic Subtype-Enriched Gene Expression Document Interindividual Differences in Their Differentiation Cascade

Cell-type origin is one of the factors that determine molecular features of tumors, but resources to validate this concept are scarce because of technical difficulties in propagating major cell types of adult organs. Previous attempts to generate such resources to study breast cancer have yielded predominantly basal-type cell lines. We have created a panel of immortalized cell lines from core breast biopsies of ancestry-mapped healthy women that form ductal structures similar to normal breast in 3D cultures and expressed markers of major cell types, including the luminal-differentiated cell-enriched ERα-FOXA1-GATA3 transcription factor network. We have also created cell lines from PROCR (CD201)+/EpCAM- cells that are likely the "normal" counterpart of the claudin-low subtype of breast cancers. RNA-seq and PAM50-intrinsic subtype clustering identified these cell lines as the "normal" counterparts of luminal A, basal, and normal-like subtypes and validated via immunostaining with basal-enriched KRT14 and luminal-enriched KRT19. We further characterized these cell lines by flow cytometry for distribution patterns of stem/basal, luminal-progenitor, mature/differentiated, multipotent PROCR+ cells, and organogenesis-enriched epithelial/mesenchymal hybrid cells using CD44/CD24, CD49f/EpCAM, CD271/EpCAM, CD201/EpCAM, and ALDEFLUOR assays and E-cadherin/vimentin double staining. These cell lines showed interindividual heterogeneity in stemness/differentiation capabilities and baseline activity of signaling molecules such as NF-κB, AKT2, pERK, and BRD4. These resources can be used to test the emerging concept that genetic variations in regulatory regions contribute to widespread differences in gene expression in "normal" conditions among the general population and can delineate the impact of cell-type origin on tumor progression.Significance: In addition to providing a valuable resource for the breast cancer research community to investigate cell-type origin of different subtypes of breast cancer, this study highlights interindividual differences in normal breast, emphasizing the need to use "normal" cells from multiple sources as controls to decipher the effects of cancer-specific genomic aberrations

Individualized breast cancer characterization through single cell analysis of tumor and adjacent-normal cells

There is a need to individualize assays for tumor molecular phenotyping, given variations in the differentiation status of tumor and normal tissues in different patients. To address this, we performed single-cell genomics of breast tumors and adjacent normal cells propagated for a short duration under growth conditions that enable epithelial reprogramming. Cells analyzed were either unselected for a specific subpopulation or phenotypically defined as undifferentiated and highly clonogenic ALDH+/CD49f+/EpCAM+ luminal progenitors, which express both basal cell and luminal cell-enriched genes. We analyzed 420 tumor cells and 284 adjacent normal cells for expression of 93 genes that included a PAM50 intrinsic subtype classifier and stemness-related genes. ALDH+/CD49f+/EpCAM+ tumor and normal cells clustered differently compared to unselected tumor and normal cells. PAM50 gene-set analyses of ALDH+/CD49f+/EpCAM+ populations efficiently identified major and minor clones of tumor cells, with the major clone resembling clinical parameters of the tumor. Similarly, a stemness-associated gene set identified clones with divergent stemness pathway activation within the same tumor. This refined expression profiling technique distinguished genes truly deregulated in cancer from genes that identify cellular precursors of tumors. Collectively, the assays presented here enable more precise identification of cancer-deregulated genes, allow for early identification of therapeutically targetable tumor cell subpopulations, and ultimately provide a refinement of precision therapeutics for cancer treatment

Dual TGFβ/BMP Pathway Inhibition Enables Expansion and Characterization of Multiple Epithelial Cell Types of the Normal and Cancerous Breast

Functional modeling of normal breast epithelial hierarchy and stromal-epithelial cell interactions have been difficult due to inability to obtain sufficient stem-progenitor-mature epithelial and stromal cells. Recently reported epithelial reprogramming assay has partially overcome this limitation, but cross contamination of cells from the feeder layer is a concern. The purpose of this study was to develop a feeder-layer independent inexpensive method to propagate multiple cell types from limited tissue resources. Cells obtained after enzymatic digestion of tissues collected at surgery or by core-needle biopsies were plated on tissue culture dishes pre-coated with laminin-5-rich conditioned media from the rat bladder tumor cell line 804G and a defined growth media with inhibitors of ROCK, TGFβ, and BMP signaling. Cells were characterized by flow cytometry, mammosphere assay, 3D cultures, and xenograft studies. Cells from the healthy breasts included CD10+/EpCAM- basal/myoepithelial, CD49f+/EpCAM+ luminal progenitor, CD49f-/EpCAM+ mature luminal, CD73+/EpCAM+/CD90- rare endogenous pluripotent somatic stem, CD73+/CD90+/EpCAM-, Estrogen Receptor alpha (ERα)-expressing ALCAM (CD166)+/EpCAM+, and ALDFLUOR+ stem/luminal progenitor subpopulations. Epithelial cells were luminal (KRT19+), basal (KRT14+) or dual positive luminal/basal hybrid cells. While breast cells derived from BRCA1, BRCA2, and PALB2 mutation carriers did not display unique characteristics, cells from women with breast cancer protective alleles showed enhanced differentiation. Cells could also be propagated from primary tumors and metastasis of breast, ovarian, and pancreatic cancer-neuroendocrine subtype. Xenograft studies confirmed tumorigenic properties of tumor-derived cells

Dependence receptor UNC5A restricts luminal to basal breast cancer plasticity and metastasis

BACKGROUND: The majority of estrogen receptor-positive (ERα+) breast cancers respond to endocrine therapies. However, resistance to endocrine therapies is common in 30% of cases, which may be due to altered ERα signaling and/or enhanced plasticity of cancer cells leading to breast cancer subtype conversion. The mechanisms leading to enhanced plasticity of ERα-positive cancer cells are unknown. METHODS: We used short hairpin (sh)RNA and/or the CRISPR/Cas9 system to knockdown the expression of the dependence receptor UNC5A in ERα+ MCF7 and T-47D cell lines. RNA-seq, quantitative reverse transcription polymerase chain reaction, chromatin immunoprecipitation, and Western blotting were used to measure the effect of UNC5A knockdown on basal and estradiol (E2)-regulated gene expression. Mammosphere assay, flow cytometry, and immunofluorescence were used to determine the role of UNC5A in restricting plasticity. Xenograft models were used to measure the effect of UNC5A knockdown on tumor growth and metastasis. Tissue microarray and immunohistochemistry were utilized to determine the prognostic value of UNC5A in breast cancer. Log-rank test, one-way, and two-way analysis of variance (ANOVA) were used for statistical analyses. RESULTS: Knockdown of the E2-inducible UNC5A resulted in altered basal gene expression affecting plasma membrane integrity and ERα signaling, as evident from ligand-independent activity of ERα, altered turnover of phosphorylated ERα, unique E2-dependent expression of genes effecting histone demethylase activity, enhanced upregulation of E2-inducible genes such as BCL2, and E2-independent tumorigenesis accompanied by multiorgan metastases. UNC5A depletion led to the appearance of a luminal/basal hybrid phenotype supported by elevated expression of basal/stem cell-enriched ∆Np63, CD44, CD49f, epidermal growth factor receptor (EGFR), and the lymphatic vessel permeability factor NTN4, but lower expression of luminal/alveolar differentiation-associated ELF5 while maintaining functional ERα. In addition, UNC5A-depleted cells acquired bipotent luminal progenitor characteristics based on KRT14+/KRT19+ and CD49f+/EpCAM+ phenotype. Consistent with in vitro results, UNC5A expression negatively correlated with EGFR expression in breast tumors, and lower expression of UNC5A, particularly in ERα+/PR+/HER2- tumors, was associated with poor outcome. CONCLUSION: These studies reveal an unexpected role of the axon guidance receptor UNC5A in fine-tuning ERα and EGFR signaling and the luminal progenitor status of hormone-sensitive breast cancers. Furthermore, UNC5A knockdown cells provide an ideal model system to investigate metastasis of ERα+ breast cancers

Pharmacological Dual Inhibition of Tumor and Tumor-Induced Functional Limitations in a Transgenic Model of Breast Cancer

Breast cancer progression is associated with systemic effects, including functional limitations and sarcopenia without the appearance of overt cachexia. Autocrine/paracrine actions of cytokines/chemokines produced by cancer cells mediate cancer progression and functional limitations. The cytokine-inducible transcription factor NF-κB could be central to this process, as it displays oncogenic functions and is integral to the Pax7:MyoD:Pgc-1β:miR-486 myogenesis axis. We tested this possibility using the MMTV-PyMT transgenic mammary tumor model and the NF-κB inhibitor dimethylaminoparthenolide (DMAPT). We observed deteriorating physical and functional conditions in PyMT+ mice with disease progression. Compared with wild-type mice, tumor-bearing PyMT+ mice showed decreased fat mass, impaired rotarod performance, and reduced grip strength as well as increased extracellular matrix (ECM) deposition in muscle. Contrary to acute cachexia models described in the literature, mammary tumor progression was associated with reduction in skeletal muscle stem/satellite-specific transcription factor Pax7. Additionally, we observed tumor-induced reduction in Pgc-1β in muscle, which controls mitochondrial biogenesis. DMAPT treatment starting at 6 to 8 weeks age prior to mammary tumor occurrence delayed mammary tumor onset and tumor growth rates without affecting metastasis. DMAPT overcame cancer-induced functional limitations and improved survival, which was accompanied with restoration of Pax7, Pgc-1β, and mitochondria levels and reduced ECM levels in skeletal muscles. In addition, DMAPT restored circulating levels of 6 out of 13 cancer-associated cytokines/chemokines changes to levels seen in healthy animals. These results reveal a pharmacological approach for overcoming cancer-induced functional limitations, and the above-noted cancer/drug-induced changes in muscle gene expression could be utilized as biomarkers of functional limitations

A priori activation of apoptosis pathways of tumor (AAAPT) technology: Development of targeted apoptosis initiators for cancer treatment

Cancer cells develop tactics to circumvent the interventions by desensitizing themselves to interventions. Amongst many, the principle routes of desensitization include a) activation of survival pathways (e.g. NF-kB, PARP) and b) downregulation of cell death pathways (e.g. CD95/CD95L). As a result, it requires high therapeutic dose to achieve tumor regression which, in turn damages normal cells through the collateral effects. Methods are needed to sensitize the low and non-responsive resistant tumor cells including cancer stem cells (CSCs) in order to evoke a better response from the current treatments. Current treatments including chemotherapy can induce cell death only in bulk cancer cells sparing CSCs and cancer resistant cells (CRCs) which are shown to be responsible for high recurrence of disease and low patient survival. Here, we report several novel tumor targeted sensitizers derived from the natural Vitamin E analogue (AMP-001-003). The drug design is based on a novel concept A priori activation of apoptosis pathways of tumor technology (AAAPT) which is designed to activate specific cell death pathways and inhibit survival pathways simultaneously and selectively in cancer cells sparing normal cells. Our results indicate that AMP-001-003 sensitize various types of cancer cells including MDA-MB-231 (triple negative breast cancer), PC3 (prostate cancer) and A543 (lung cancer) cells resulting in reducing the IC-50 of doxorubicin in vitro when used as a combination. At higher doses, AMP-001 acts as an anti-tumor agent on its own. The synergy between AMP-001 and doxorubicin could pave a new pathway to use AAAPT leading molecules as neoadjuvant to chemotherapy to achieve better efficacy and reduced off-target toxicity compared to the current treatments

Distinct Effects of Adipose-derived Stem Cells and Adipocytes on Normal and Cancer Cell Hierarchy

Adipose-derived stem cells (ASC) have received considerable attention in oncology because of the known direct link between obesity and cancer as well as the use of ASCs in reconstructive surgery after tumor ablation. Previous studies have documented how cancer cells commandeer ASCs to support their survival by altering extracellular matrix composition and stiffness, migration, and metastasis. This study focused on delineating the effects of ASCs and adipocytes on the self-renewal of stem/progenitor cells and hierarchy of breast epithelial cells. The immortalized breast epithelial cell line MCF10A, ductal carcinoma in situ (DCIS) cell lines MCF10DCIS.com and SUM225, and MCF10A-overexpressing SRC oncogene were examined using a mammosphere assay and flow cytometry for the effects of ASCs on their self-renewal and stem-luminal progenitor-differentiated cell surface marker profiles. Interestingly, ASCs promoted the self-renewal of all cell types except SUM225. ASC coculture or treatment with ASC conditioned media altered the number of CD49fhigh/EpCAMlow basal/stem-like and CD49fmedium/EpCAMmedium luminal progenitor cells. Among multiple factors secreted by ASCs, IFNγ and hepatocyte growth factor (HGF) displayed unique actions on epithelial cell hierarchy. IFNγ increased stem/progenitor-like cells while simultaneously reducing the size of mammospheres, whereas HGF increased the size of mammospheres with an accompanying increase in luminal progenitor cells. ASCs expressed higher levels of HGF, whereas adipocytes expressed higher levels of IFNγ. As luminal progenitor cells are believed to be prone for transformation, IFNγ and HGF expression status of ASCs may influence susceptibility for developing breast cancer as well as on outcomes of autologous fat transplantation on residual/dormant tumor cells. Implications: This study suggests that the ratio of ASCs to adipocytes influences cancer cell hierarchy, which may impact incidence and progression