Oxygen tension-dependent variability in the cancer cell kinome impacts signaling pathways and response to targeted therapies.

Most cells in solid tumors are exposed to oxygen levels between 0.5% and 5%. We developed an approach that allows collection, processing, and evaluation of cancer and non-cancer cells under physioxia, while preventing exposure to ambient air. This aided comparison of baseline and drug-induced changes in signaling pathways under physioxia and ambient oxygen. Using tumor cells from transgenic models of breast cancer and cells from breast tissues of clinically breast cancer-free women, we demonstrate oxygen-dependent differences in cell preference for epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor beta (PDGFRß) signaling. Physioxia caused PDGFRß-mediated activation of AKT and extracellular regulated kinase (ERK) that reduced sensitivity to EGFR and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) inhibition and maintained PDGFRß+ epithelial-mesenchymal hybrid cells with potential cancer stem cell (CSC) properties. Cells in ambient air displayed differential EGFR activation and were more sensitive to targeted therapies. Our data emphasize the importance of oxygen considerations in preclinical cancer research to identify effective drug targets and develop combination therapy regimens.

Signaling Pathway Alterations Driven by BRCA1 and BRCA2 Germline Mutations are Sufficient to Initiate Breast Tumorigenesis by the PIK3CAH1047R Oncogene.

Single-cell transcriptomics studies have begun to identify breast epithelial cell and stromal cell specific transcriptome differences between BRCA1/2 mutation carriers and non-carriers. We generated a single-cell transcriptome atlas of breast tissues from BRCA1, BRCA2 mutation carriers and compared this single-cell atlas of mutation carriers with our previously described single-cell breast atlas of healthy non-carriers. We observed that BRCA1 but not BRCA2 mutations altered the ratio between basal (basal-myoepithelial), luminal progenitor (luminal adaptive secretory precursor, LASP), and mature luminal (luminal hormone sensing) cells in breast tissues. A unique subcluster of cells within LASP cells is underrepresented in case of BRCA1 and BRCA2 mutation carriers compared with non-carriers. Both BRCA1 and BRCA2 mutations specifically altered transcriptomes in epithelial cells which are an integral part of NFκB, LARP1, and MYC signaling. Signaling pathway alterations in epithelial cells unique to BRCA1 mutations included STAT3, BRD4, SMARCA4, HIF2A/EPAS1, and Inhibin A signaling. BRCA2 mutations were associated with upregulation of IL6, PDK1, FOXO3, and TNFSF11 signaling. These signaling pathway alterations are sufficient to alter sensitivity of BRCA1/BRCA2-mutant breast epithelial cells to transformation as epithelial cells from BRCA1 mutation carriers overexpressing hTERT + PIK3CAH1047R generated adenocarcinomas, whereas similarly modified mutant BRCA2 cells generated basal carcinomas in NSG mice. Thus, our studies provide a high-resolution transcriptome atlas of breast epithelial cells of BRCA1 and BRCA2 mutation carriers and reveal their susceptibility to PIK3CA mutation-driven transformation. SIGNIFICANCE: This study provides a single-cell atlas of breast tissues of BRCA1/2 mutation carriers and demonstrates that aberrant signaling due to BRCA1/2 mutations is sufficient to initiate breast cancer by mutant PIK3CA.

Stromal heterogeneity may explain increased incidence of metaplastic breast cancer in women of African descent.

The biologic basis of genetic ancestry-dependent variability in disease incidence and outcome is just beginning to be explored. We recently reported enrichment of a population of ZEB1-expressing cells located adjacent to ductal epithelial cells in normal breasts of women of African ancestry compared to those of European ancestry. In this study, we demonstrate that these cells have properties of fibroadipogenic/mesenchymal stromal cells that express PROCR and PDGFRα and transdifferentiate into adipogenic and osteogenic lineages. PROCR + /ZEB1 + /PDGFRα+ (PZP) cells are enriched in normal breast tissues of women of African compared to European ancestry. PZP: epithelial cell communication results in luminal epithelial cells acquiring basal cell characteristics and IL-6-dependent increase in STAT3 phosphorylation. Furthermore, level of phospho-STAT3 is higher in normal and cancerous breast tissues of women of African ancestry. PZP cells transformed with HRasG12V ± SV40-T/t antigens generate metaplastic carcinoma suggesting that these cells are one of the cells-of-origin of metaplastic breast cancers.

TONSL Is an Immortalizing Oncogene and a Therapeutic Target in Breast Cancer.

Study of genomic aberrations leading to immortalization of epithelial cells has been technically challenging due to the lack of isogenic models. To address this, we used healthy primary breast luminal epithelial cells of different genetic ancestry and their hTERT-immortalized counterparts to identify transcriptomic changes associated with immortalization. Elevated expression of TONSL (Tonsoku-like, DNA repair protein) was identified as one of the earliest events during immortalization. TONSL, which is located on chromosome 8q24.3, was found to be amplified in approximately 20% of breast cancers. TONSL alone immortalized primary breast epithelial cells and increased telomerase activity, but overexpression was insufficient for neoplastic transformation. However, TONSL-immortalized primary cells overexpressing defined oncogenes generated estrogen receptor-positive adenocarcinomas in mice. Analysis of a breast tumor microarray with approximately 600 tumors revealed poor overall and progression-free survival of patients with TONSL-overexpressing tumors. TONSL increased chromatin accessibility to pro-oncogenic transcription factors, including NF-κB and limited access to the tumor-suppressor p53. TONSL overexpression resulted in significant changes in the expression of genes associated with DNA repair hubs, including upregulation of several genes in the homologous recombination (HR) and Fanconi anemia pathways. Consistent with these results, TONSL-overexpressing primary cells exhibited upregulated DNA repair via HR. Moreover, TONSL was essential for growth of TONSL-amplified breast cancer cell lines in vivo, and these cells were sensitive to TONSL-FACT complex inhibitor CBL0137. Together, these findings identify TONSL as a regulator of epithelial cell immortalization to facilitate cancer initiation and as a target for breast cancer therapy. SIGNIFICANCE: The chr.8q24.3 amplicon-resident gene TONSL is upregulated during the initial steps of tumorigenesis to support neoplastic transformation by increasing DNA repair and represents a potential therapeutic target for treating breast cancer.

A human skeletal muscle stem/myotube model reveals multiple signaling targets of cancer secretome in skeletal muscle.

Skeletal muscle dysfunction or reprogramming due to the effects of the cancer secretome is observed in multiple malignancies. Although mouse models are routinely used to study skeletal muscle defects in cancer, because of species specificity of certain cytokines/chemokines in the secretome, a human model system is required. Here, we establish simplified multiple skeletal muscle stem cell lines (hMuSCs), which can be differentiated into myotubes. Using single nuclei ATAC-seq (snATAC-seq) and RNA-seq (snRNA-seq), we document chromatin accessibility and transcriptomic changes associated with the transition of hMuSCs to myotubes. Cancer secretome accelerated stem to myotube differentiation, altered the alternative splicing machinery and increased inflammatory, glucocorticoid receptor, and wound healing pathways in hMuSCs. Additionally, cancer secretome reduced metabolic and survival pathway associated miR-486, AKT, and p53 signaling in hMuSCs. hMuSCs underwent myotube differentiation when engrafted into NSG mice and thus providing a humanized in vivo skeletal muscle model system to study cancer cachexia.

Tumor collection/processing under physioxia uncovers highly relevant signaling networks and drug sensitivity.

Preclinical studies of primary cancer cells are typically done after tumors are removed from patients or animals at ambient atmospheric oxygen (O2, ~21%). However, O2 concentrations in organs are in the ~3 to 10% range, with most tumors in a hypoxic or 1 to 2% O2 environment in vivo. Although effects of O2 tension on tumor cell characteristics in vitro have been studied, these studies are done only after tumors are first collected and processed in ambient air. Similarly, sensitivity of primary cancer cells to anticancer agents is routinely examined at ambient O2. Here, we demonstrate that tumors collected, processed, and propagated at physiologic O2 compared to ambient air display distinct differences in key signaling networks including LGR5/WNT, YAP, and NRF2/KEAP1, nuclear reactive oxygen species, alternative splicing, and sensitivity to targeted therapies. Therefore, evaluating cancer cells under physioxia could more closely recapitulate their physiopathologic status in the in vivo microenvironment.

Acquisition, processing, and single-cell analysis of normal human breast tissues from a biobank.

The Komen Tissue Bank is the only biorepository in the world for normal breast tissues from women. Below we report the acquisition and processing of breast tissue from volunteer donors and describe an experimental and analysis pipeline to generate a single-cell atlas. This atlas is based on single-cell RNA-seq and is useful to derive breast epithelial cell subcluster-specific gene expression signatures, which can be applied to breast cancer gene expression data to identify putative cell-of-origin. For complete details on the use and execution of this protocol, please refer to Bhat-Nakshatri et al. (2021).

Bidirectional Regulatory Cross-Talk between Cell Context and Genomic Aberrations Shapes Breast Tumorigenesis.

Breast cancers are classified into five intrinsic subtypes and 10 integrative clusters based on gene expression patterns and genomic aberrations, respectively. Although the cell-of-origin, adaptive plasticity, and genomic aberrations shape dynamic transcriptomic landscape during cancer progression, how interplay between these three core elements governs obligatory steps for a productive cancer progression is unknown. Here, we used genetic ancestry-mapped immortalized breast epithelial cell lines generated from breast biopsies of healthy women that share gene expression profiles of luminal A, normal-like, and basal-like intrinsic subtypes of breast cancers and breast cancer relevant oncogenes to develop breast cancer progression model. Using flow cytometry, mammosphere growth, signaling pathway, DNA damage response, and in vivo tumorigenicity assays, we provide evidence that establishes cell context-dependent effects of oncogenes in conferring plasticity, self-renewal/differentiation, intratumor heterogeneity, and metastatic properties. In contrast, oncogenic aberrations, independent of cell context, shaped response to DNA damage-inducing agents. Collectively, this study reveals how the same set of genomic aberration can have distinct effects on tumor characteristics based on cell-of-origin of tumor and highlights the need to utilize multiple "normal" epithelial cell types to decipher oncogenic properties of a gene of interest. In addition, by creating multiple isogenic cell lines ranging from primary cells to metastatic variants, we provide resources to elucidate cell-intrinsic properties and cell-oncogene interactions at various stages of cancer progression. IMPLICATIONS: Our findings demonstrate that how an interplay between the normal cell type that encountered genomic aberrations and type of genomic aberration influences heterogeneity, self-renewal/differentiation, and tumor properties including propensity for metastasis.

Antitumor properties of novel sesquiterpene lactone analogs as NFκB inhibitors that bind to the IKKß ubiquitin-like domain (ULD).

Melampomagnolide B (MMB, 3) is a parthenolide (PTL, 1) based sesquiterpene lactone that has been used as a template for the synthesis of a plethora of lead anticancer agents owing to its reactive C-10 primary hydroxyl group. Such compounds have been shown to inhibit the IKKß subunit, preventing phosphorylation of the cytoplasmic IκB inhibitory complex. The present study focuses on the synthesis and in vitro antitumor properties of novel benzyl and phenethyl carbamates of MMB (7a-7k). Screening of these MMB carbamates identified analogs with potent growth inhibition properties against a panel of 60 human cancer cell lines (71% of the molecules screened had GI50 values < 2 µM). Two analogs, the benzyl carbamate 7b and the phenethyl carbamate7k, were the most active compounds. Lead compound 7b inhibited cell proliferation in M9 ENL AML cells, and in TMD-231, OV-MD-231 and SUM149 breast cancer cell lines. Interestingly, mechanistic studies showed that 7b did not inhibit p65 phosphorylation in M9 ENL AML and OV-MD-231 cells, but did inhibit phophorylation of both p65 and IκBα in SUM149 cells. 7b also reduced NFκB binding to DNA in both OV-MD-231 and SUM149 cells. Molecular docking studies indicated that 7b and 7k are both predicted to interact with the ubiquitin-like domain (ULD) of the IKKß subunit. These data suggest that in SUM149 cells, 7b is likely acting as an allosteric inhibitor of IKKß, whereas in M9 ENL AML and OV-MD-231 cells 7b is able to inhibit an event after IκB/p65/p50 phosphorylation by IKKß that leads to inhibition of NFκB activation and reduction in NFκB-DNA binding. Analog 7b was by far the most potent compound in either carbamate series, and was considered an important lead compound for further optimization and development as an anticancer agent.

Hormonally Regulated Myogenic miR-486 Influences Sex-specific Differences in Cancer-induced Skeletal Muscle Defects.

Cancer-induced skeletal muscle defects show sex-specific differences in severity with men performing poorly compared to women. Hormones and sex chromosomal differences are suggested to mediate these differences, but the functional skeletal muscle markers to document these differences are unknown. We show that the myogenic microRNA miR-486 is a marker of sex-specific differences in cancer-induced skeletal muscle defects. Cancer-induced loss of circulating miR-486 was more severe in men with bladder, lung, and pancreatic cancers compared to women with the same cancer types. In a syngeneic model of pancreatic cancer, circulating and skeletal muscle loss of miR-486 was more severe in male mice compared to female mice. Estradiol (E2) and the clinically used selective estrogen receptor modulator toremifene increased miR-486 in undifferentiated and differentiated myoblast cell line C2C12 and E2-inducible expression correlated with direct binding of estrogen receptor alpha (ERα) to the regulatory region of the miR-486 gene. E2 and toremifene reduced the actions of cytokines such as myostatin, transforming growth factor ß, and tumor necrosis factor α, which mediate cancer-induced skeletal muscle wasting. E2- and toremifene-treated C2C12 myoblast/myotube cells contained elevated levels of active protein kinase B (AKT) with a corresponding decrease in the levels of its negative regulator PTEN, which is a target of miR-486. We propose an ERα:E2-miR-486-AKT signaling axis, which reduces the deleterious effects of cancer-induced cytokines/chemokines on skeletal muscle mass and/or function.

Aging-associated skeletal muscle defects in HER2/Neu transgenic mammary tumor model.

BACKGROUND: Loss of skeletal muscle volume and resulting in functional limitations are poor prognostic markers in breast cancer patients. Several molecular defects in skeletal muscle including reduced MyoD levels and increased protein turn over due to enhanced proteosomal activity have been suggested as causes of skeletal muscle loss in cancer patients. However, it is unknown whether molecular defects in skeletal muscle are dependent on tumor etiology. METHODS: We characterized functional and molecular defects of skeletal muscle in MMTV-Neu (Neu+) mice (n= 6-12), an animal model that represents HER2+ human breast cancer, and compared the results with well-characterized luminal B breast cancer model MMTV-PyMT (PyMT+). Functional studies such as grip strength, rotarod performance, and ex vivo muscle contraction were performed to measure the effects of cancer on skeletal muscle. Expression of muscle-enriched genes and microRNAs as well as circulating cytokines/chemokines were measured. Since NF-κB pathway plays a significant role in skeletal muscle defects, the ability of NF-κB inhibitor dimethylaminoparthenolide (DMAPT) to reverse skeletal muscle defects was examined. RESULTS: Neu+ mice showed skeletal muscle defects similar to accelerated aging. Compared to age and sex-matched wild type mice, Neu+ tumor-bearing mice had lower grip strength (202±6.9 vs. 179±6.8 g grip force, p=0.0069) and impaired rotarod performance (108±12.1 vs. 30±3.9 seconds, P<0.0001), which was consistent with reduced muscle contractibility (p<0.0001). Skeletal muscle of Neu+ mice (n=6) contained lower levels of CD82+ (16.2±2.9 vs 9.0±1.6) and CD54+ (3.8±0.5 vs 2.4±0.4) muscle stem and progenitor cells (p<0.05), suggesting impaired capacity of muscle regeneration, which was accompanied by decreased MyoD, p53 and miR-486 expression in muscles (p<0.05). Unlike PyMT+ mice, which showed skeletal muscle mitochondrial defects including reduced mitochondria levels and Pgc1ß, Neu+ mice displayed accelerated aging-associated changes including muscle fiber shrinkage and increased extracellular matrix deposition. Circulating "aging factor" and cachexia and fibromyalgia-associated chemokine Ccl11 was elevated in Neu+ mice (1439.56±514 vs. 1950±345 pg/ml, p<0.05). Treatment of Neu+ mice with DMAPT significantly restored grip strength (205±6 g force), rotarod performance (74±8.5 seconds), reversed molecular alterations associated with skeletal muscle aging, reduced circulating Ccl11 (1083.26 ±478 pg/ml), and improved animal survival. CONCLUSIONS: These results suggest that breast cancer subtype has a specific impact on the type of molecular and structure changes in skeletal muscle, which needs to be taken into consideration while designing therapies to reduce breast cancer-induced skeletal muscle loss and functional limitations.

A single-cell atlas of the healthy breast tissues reveals clinically relevant clusters of breast epithelial cells.

Single-cell RNA sequencing (scRNA-seq) is an evolving technology used to elucidate the cellular architecture of adult organs. Previous scRNA-seq on breast tissue utilized reduction mammoplasty samples, which are often histologically abnormal. We report a rapid tissue collection/processing protocol to perform scRNA-seq of breast biopsies of healthy women and identify 23 breast epithelial cell clusters. Putative cell-of-origin signatures derived from these clusters are applied to analyze transcriptomes of ~3,000 breast cancers. Gene signatures derived from mature luminal cell clusters are enriched in ~68% of breast cancers, whereas a signature from a luminal progenitor cluster is enriched in ~20% of breast cancers. Overexpression of luminal progenitor cluster-derived signatures in HER2+, but not in other subtypes, is associated with unfavorable outcome. We identify TBX3 and PDK4 as genes co-expressed with estrogen receptor (ER) in the normal breasts, and their expression analyses in >550 breast cancers enable prognostically relevant subclassification of ER+ breast cancers.

Nonlinear relationship between chromatin accessibility and estradiol-regulated gene expression.

Chromatin accessibility is central to basal and inducible gene expression. Through ATAC-seq experiments in estrogen receptor-positive (ER+) breast cancer cell line MCF-7 and integration with multi-omics data, we found estradiol (E2) induced chromatin accessibility changes in a small number of breast cancer-relevant E2-regulated genes. As expected, open chromatin regions associated with E2-inducible gene expression showed enrichment of estrogen response element (ERE) and those associated with E2-repressible gene expression were enriched for ERE, PBX1, and PBX3. While a significant number of open chromatin regions showed pioneer factor FOXA1 occupancy in the absence of E2, E2-treatment further enhanced FOXA1 occupancy suggesting that ER-E2 enhances chromatin occupancy of FOXA1 to a subset of E2-regulated genes. Surprisingly, promoters of 80% and enhancers of 60% of E2-inducible genes displayed closed chromatin configuration both in the absence and presence of E2. Integration of ATAC-seq data with ERα ChIP-seq data revealed that ~40% ERα binding sites in the genome are found in chromatin regions that are not accessible as per ATAC-seq. Such ERα binding regions were enriched for binding sites of multiple nuclear receptors including ER, ESRRB, ERRγ, COUP-TFII (NR2F2), RARα, EAR2 as well as traditional pioneer factors FOXA1 and GATA3. Similar data were also obtained when ERα ChIP-seq data were integrated with MNase-seq and DNase-seq data sets. In summation, our results reveal complex mechanisms of ER-E2 interaction with nucleosomes. Notably, "closed chromatin" configuration as defined by ATAC-seq or by other techniques is not necessarily associated with lack of gene expression and technical limitations may preclude ATAC-seq to demonstrate accessibility of chromatin regions that are bound by ERα.

Breast Cancer Cell Detection and Characterization from Breast Milk-Derived Cells.

Radiologic techniques remain the main method for early detection for breast cancer and are critical to achieve a favorable outcome from cancer. However, more sensitive detection methods to complement radiologic techniques are needed to enhance early detection and treatment strategies. Using our recently established culturing method that allows propagation of normal and cancerous breast epithelial cells of luminal origin, flow cytometry characterization, and genomic sequencing, we show that cancer cells can be detected in breast milk. Cells derived from milk from the breast with cancer were enriched for CD49f+/EpCAM-, CD44+/CD24-, and CD271+ cancer stem-like cells (CSC). These CSCs carried mutations within the cytoplasmic retention domain of HDAC6, stop/gain insertion in MORF4L1, and deletion mutations within SWI/SNF complex component SMARCC2. CSCs were sensitive to HDAC6 inhibitors, BET bromodomain inhibitors, and EZH2 inhibitors, as mutations in SWI/SNF complex components are known to increase sensitivity to these drugs. Among cells derived from breast milk of additional ten women not known to have breast cancer, two of them contained cells that were enriched for the CSC phenotype and carried mutations in NF1 or KMT2D, which are frequently mutated in breast cancer. Breast milk-derived cells with NF1 mutations also carried copy-number variations in CDKN2C, PTEN, and REL genes. The approach described here may enable rapid cancer cell characterization including driver mutation detection and therapeutic screening for pregnancy/postpartum breast cancers. Furthermore, this method can be developed as a surveillance or early detection tool for women at high risk for developing breast cancer. SIGNIFICANCE: These findings describe how a simple method for characterization of cancer cells in pregnancy and postpartum breast cancer can be exploited as a surveillance tool for women at risk of developing breast cancer.

Dual TGFß/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 and 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 precoated with laminin-5-rich-conditioned media from the rat bladder tumor cell line 804G and a defined growth media with inhibitors of ROCK, TGFß, 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-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. IMPLICATIONS: Our method expands the scope of individualized studies of patient-derived cells and provides resources to model epithelial-stromal interactions under normal and pathologic conditions.

MMB triazole analogs are potent NF-κB inhibitors and anti-cancer agents against both hematological and solid tumor cells.

Triazole derivatives of melampomagnolide B (MMB) have been synthesized via click chemistry methodologies and screened against a panel of 60 human cancer cell lines. Several derivatives showed promising anti-cancer activity, affording growth inhibition (GI50) values in the nanomolar range (GI50 = 0.02-0.99 µM). Lead compound 7h exhibited EC50 values of 400 nM and 700 nM, respectively, against two AML clinical specimens. Compound 7h was significantly more potent than parthenolide as an inhibitor of p65 phosphorylation in both hematological and solid tumor cell lines, indicating its ability to inhibit the NF-κB pathway. In TMD-231 breast cancer cells, treatment with 7h reduced DNA binding activity of NF-κB through inhibition of IKK-ß mediated p65 phosphorylation and caused elevation of basal IκBα levels through inhibition of constitutive IκBα turnover and NF-κB activation. Molecular docking and dynamic modeling studies indicated that 7h interacts with the kinase domain of the monomeric IKKß subunit, leading to inhibition of IKKß activation, and compromising phosphorylation of downstream targets of the NF-κB pathway; dynamic modeling studies show that this interaction also causes unwinding of the α-helix of the NEMO binding site on IKKß. Molecular docking studies with 10, a water-soluble analog of 7h, demonstrate that this analog interacts with the dimerization/oligomerization domain of monomeric IKKß and may inhibit oligomer formation and subsequent autophosphorylation. Sesquiterpene lactones 7h and 10 are considered ideal candidates for potential clinical development.

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. Cancer Res; 78(17); 5107-23. ©2018 AACR.

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.

Mutational landscape of RNA-binding proteins in human cancers.

RNA Binding Proteins (RBPs) are a class of post-transcriptional regulatory molecules which are increasingly documented to be dysfunctional in cancer genomes. However, our current understanding of these alterations is limited. Here, we delineate the mutational landscape of ∼1300 RBPs in ∼6000 cancer genomes. Our analysis revealed that RBPs have an average of ∼3 mutations per Mb across 26 cancer types. We identified 281 RBPs to be enriched for mutations (GEMs) in at least one cancer type. GEM RBPs were found to undergo frequent frameshift and inframe deletions as well as missense, nonsense and silent mutations when compared to those that are not enriched for mutations. Functional analysis of these RBPs revealed the enrichment of pathways associated with apoptosis, splicing and translation. Using the OncodriveFM framework, we also identified more than 200 candidate driver RBPs that were found to accumulate functionally impactful mutations in at least one cancer. Expression levels of 15% of these driver RBPs exhibited significant difference, when transcriptome groups with and without deleterious mutations were compared. Functional interaction network of the driver RBPs revealed the enrichment of spliceosomal machinery, suggesting a plausible mechanism for tumorogenesis while network analysis of the protein interactions between RBPs unambiguously revealed the higher degree, betweenness and closeness centrality for driver RBPs compared to non-drivers. Analysis to reveal cancer-specific Ribonucleoprotein (RNP) mutational hotspots showed extensive rewiring even among common drivers between cancer types. Knockdown experiments on pan-cancer drivers such as SF3B1 and PRPF8 in breast cancer cell lines, revealed cancer subtype specific functions like selective stem cell features, indicating a plausible means for RBPs to mediate cancer-specific phenotypes. Hence, this study would form a foundation to uncover the contribution of the mutational spectrum of RBPs in dysregulating the post-transcriptional regulatory networks in different cancer types.

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-1ß: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-1ß 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-1ß, 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. Mol Cancer Ther; 16(12); 2747-58. ©2017 AACR.