Luminal Breast Cancer Cells Research Articles

Hippo-TAZ signaling is the master regulator of the onset of triple-negative basal-like breast cancers.

Breast cancer is the most frequent malignancy in women worldwide. Basal-like breast cancer (BLBC) is the most aggressive form of this disease, and patients have a poor prognosis. Here, we present data suggesting that the Hippo-transcriptional coactivator with PDZ-binding motif (TAZ) pathway is a key driver of BLBC onset and progression. Deletion of Mob1a/b in mouse mammary luminal epithelium induced rapid and highly reproducible mammary tumorigenesis that was dependent on TAZ but not yes-associated protein 1 (YAP1). In situ early-stage BLBC-like malignancies developed in mutant animals by 2 wk of age, and invasive BLBC appeared by 4 wk. In a human estrogen receptor+ luminal breast cancer cell line, TAZ hyperactivation skewed the features of these luminal cells to the basal phenotype, consistent with the aberrant TAZ activation frequently observed in human precancerous BLBC lesions. TP53 mutation is rare in human precancerous BLBC but frequent in invasive BLBC. Addition of Trp53 deficiency to our Mob1a/b-deficient mouse model enhanced tumor grade and accelerated cancer progression. Our work justifies targeting the Hippo-TAZ pathway as a therapy for human BLBC, and our mouse model represents a powerful tool for evaluating candidate agents.

The NFAT3/RERG Complex in Luminal Breast Cancers Is Required to Inhibit Cell Invasion and May Be Correlated With an Absence of Axillary Lymph Nodes Colonization.

Luminal breast cancers represent 70% of newly diagnosed breast cancers per annum and have a relatively good prognosis compared with triple-negative breast cancers. Luminal tumors that are responsive to hormonal therapy are particularly associated with a favorable prognosis. Nonetheless, the absolute number of metastatic relapses in luminal cancers is larger than in triple-negative breast cancers. A better understanding of the biology of luminal cancers, control of metastases formation, and identification of predictive markers of their evolution are therefore still necessary. In this context, we previously disclosed the key role of NFAT3 in regulating luminal breast cancer invasion. We have now identified a specific inhibitory region, in the C-terminal part of NFAT3, required for the inhibition of invasion of the human luminal breast cancer cell line T-47D. Indeed, we showed that this 85 amino acid C-terminal region acts as a dominant negative form of NFAT3 and that its overexpression in the T-47D cell line led to increased cell invasion. Mechanistically, we have revealed that this region of NFAT3 interacts with the small Ras GTPase RERG (RAS like estrogen regulated growth inhibitor) and shown that RERG expression is required for NFAT3 to impede T-47D cell invasion. We have validated the association of NFAT3 with RERG in human luminal breast cancer tissues. We have shown an increase of the quantity of the NFAT3/RERG complexes in patients without axillary lymph node colonization and therefore proposed that the detection of this complex may be a non-invasive marker of axillary lymph node colonization.

Abstract 2936: Multi-omic characterization of transitional cell populations in breast cancer

Abstract Breast cancer is a heterogeneous collection of diseases grouped by hormone receptor status or by expression of key subtype-determining genes. Breast cancer subtypes, in particular basal-like breast cancer and the luminal breast cancer subtypes, differ by hormonal receptor status, proliferation, genomic instability and mutational signatures, treatment response, and prognosis. The highly distinct signatures of basal-like and luminal breast cancer suggest that they may have different cells of origin within the breast duct. As part of the Washington University Human Tumor Atlas Network (WU-HTAN) program, we generated multi-omic data for 53 samples from 37 breast cancer tumors and 4 normal adjacent tissues. Genomic subtyping was applied to both bulk and single-nucleus RNA sequencing. Analysis of single-nucleus gene expression and chromatin accessibility in epithelial cells underscores similarities between basal-like breast cancer and luminal progenitor cells within the breast duct, and between luminal breast cancer and mature luminal ductal cells. This study links distinct breast cancer subtypes to normal breast cell populations and suggests distinct cells of origin for these cancer types. Citation Format: Michael D. Iglesia, Reyka G. Jayasinghe, Daniel Cui Zhou, Nadezhda V. Terekhanova, John Herndon, Alla Karpova, Siqi Chen, Nataly Naser Al Deen, Kazuhito Sato, Feng Chen, Deborah J. Veis, Ryan C. Fields, William E. Gillanders, Li Ding. Multi-omic characterization of transitional cell populations in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2936.

FOXF2 oppositely regulates stemness in luminal and basal-like breast cancer cells through the Wnt/beta-catenin pathway

The stemness of cancer cells contributes to tumorigenesis, the heterogeneity of malignancies, cancer metastasis, and therapeutic resistance. However, the roles and regulatory mechanisms maintaining stemness among breast cancer subtypes remain elusive. Our previous studies have demonstrated that ectopic expression and dynamic alteration of the mesenchymal transcription factor forkhead box F2 (FOXF2) differentially regulates breast cancer progression and metastasis organotropism in a cell subtype-specific manner. Here, we reveal the underlying mechanism by which FOXF2 enhances stemness in luminal breast cancer cells but suppresses that in basal-like breast cancer (BLBC) cells. We show that luminal breast cancer and BLBC cells with FOXF2-regulated stemness exhibit partial mesenchymal stem cell properties that toward osteogenic differentiation and myogenic differentiation, respectively. Furthermore, we show that FOXF2 activates the Wnt signaling pathway in luminal breast cancer cells but represses this pathway in BLBC cells by recruiting nuclear receptor coactivator 3 (NCoA3) and nuclear receptor corepressor 1 (NCoR1) to the promoters of Wnt family member 2B (WNT2B) and frizzled class receptor 1 (FZD1) genes to activate and repress their transcription, respectively. We propose that targeting the Wnt signaling pathway is a promising strategy for the treatment of breast cancers with dysregulated expression of FOXF2.

Ultrasensitive Detection of MCF-7 Cells with a Carbon Nanotube-Based Optoelectronic-Pulse Sensor Framework.

Biosensors are of vital significance for healthcare by supporting the management of infectious diseases for preventing pandemics and the diagnosis of life-threatening conditions such as cancer. However, the advancement of the field can be limited by low sensing accuracy. Here, we altered the bioelectrical signatures of the cells using carbon nanotubes (CNTs) via structural loosening effects. Using an alternating current (AC) pulse under light irradiation, we developed a photo-assisted AC pulse sensor based on CNTs to differentiate between healthy breast epithelial cells (MCF-10A) and luminal breast cancer cells (MCF-7) within a heterogeneous cell population. We observed a previously undemonstrated increase in current contrast for MCF-7 cells with CNTs compared to MCF-10A cells with CNTs under light exposure. Moreover, we obtained a detection limit of ∼1.5 × 103 cells below a baseline of ∼1 × 104 cells for existing electrical-based sensors for an adherent, heterogeneous cell population. All-atom molecular dynamics (MD) simulations reveal that interactions between the embedded CNT and cancer cell membranes result in a less rigid lipid bilayer structure, which can facilitate CNT translocation for enhancing current. This as-yet unconsidered cancer cell-specific method based on the unique optoelectrical properties of CNTs represents a strategy for unlocking the detection of a small population of cancer cells and provides a promising route for the early diagnosis, monitoring, and staging of cancer.

Three-Dimensional Organotypic Cultures Reshape the microRNAs Transcriptional Program in Breast Cancer Cells.

Simple SummaryThree-dimensional (3D) cell cultures have several advantages over conventional monolayer two-dimensional (2D) cultures as they can better mimic tumor biology. This study delineates the changes in microRNAs (miRNAs) expression patterns of breast cancer cells cultured in 3D and 2D conditions. 3D organotypic cultures showed morphological changes such as cell–cell and cell–extracellular matrix interactions associated with a loss of polarity and reorganization on bulk structures in both basal Hs578T and luminal T47D breast cancer cells. Data indicate that downregulated miRNAs in Hs578T 3D cultures, relative to the 2D condition, contribute to a positive regulation of biological processes such as response to hypoxia and focal adhesion, whereas overexpressed miRNAs were related to negative regulation of the cell cycle. Remarkably, the reprogramming of miRNAs transcriptional profile was accompanied by changes in the expression of key miRNA/mRNA coregulation networks, such as miR-935/HIF-1A, which correlated with the expression found in clinical breast tumors and predicted poor patient outcomes. These data have implications in our understanding of cancer biology and impact the miRNA/mRNA regulatory axes of cells grown in 3D cultures. Our data represent a guide for novel miRNAs candidates for functional analysis, including the response to therapy and biomarkers discovery in breast cancer.The 3D organotypic cultures, which depend on the growth of cells over the extracellular matrix (ECM) used as a scaffold, can better mimic several characteristics of solid cancers that influence tumor biology and the response to drug therapies. Most of our current knowledge on cancer is derived from studies in 2D cultures, which lack the ECM-mediated microenvironment. Moreover, the role of miRNAs that is critical for fine-tuning of gene expression is poorly understood in 3D cultures. The aim of this study was to compare the miRNA expression profiles of breast cancer cells grown in 2D and 3D conditions. On an on-top 3D cell culture model using a basement membrane matrix enriched with laminin, collagen IV, entactin, and heparin-sulfate proteoglycans, the basal B (Hs578T) and luminal (T47D) breast cancer cells formed 3D spheroid-like stellate and rounded mass structures, respectively. Morphological changes in 3D cultures were observed as cell stretching, cell–cell, and cell–ECM interactions associated with a loss of polarity and reorganization on bulk structures. Interestingly, we found prolongations of the cytoplasmic membrane of Hs578T cells similar to tunneled nanotubes contacting between neighboring cells, suggesting the existence of cellular intercommunication processes and the possibility of fusion between spheroids. Expression profiling data revealed that 354 miRNAs were differentially expressed in 3D relative to 2D cultures in Hs578T cells. Downregulated miRNAs may contribute to a positive regulation of genes involved in hypoxia, catabolic processes, and focal adhesion, whereas overexpressed miRNAs modulate genes involved in negative regulation of the cell cycle. Target genes of the top ten modulated miRNAs were selected to construct miRNA/mRNA coregulation networks. Around 502 interactions were identified for downregulated miRNAs, including miR-935/HIF1A and miR-5189-3p/AKT that could contribute to cell migration and the response to hypoxia. Furthermore, the expression levels of miR-935 and its target HIF1A correlated with the expression found in clinical tumors and predicted poor outcomes. On the other hand, 416 interactions were identified for overexpressed miRNAs, including miR-6780b-5p/ANKRD45 and miR-7641/CDK4 that may result in cell proliferation inhibition and cell cycle arrest in quiescent layers of 3D cultures. In conclusion, 3D cultures could represent a suitable model that better resembles the miRNA transcriptional programs operating in tumors, with implications not only in the understanding of basic cancer biology in 3D microenvironments, but also in the identification of novel biomarkers of disease and potential targets for personalized therapies in cancer.

Disentangling ERBB Signaling in Breast Cancer Subtypes-A Model-Based Analysis.

Simple SummaryBreast cancer subtypes are characterized by the expression and activity of estrogen-, progesterone- and HER2-receptors and differ by the treatment as well as patient prognosis. Tumors of the HER2-subtype overexpress this receptor and are successfully targeted with anti-HER2 therapies. We wanted to know if the HER2-receptor and the downstream signaling network act similarly also in the other subtypes and if this network could potentially be a therapeutic target beyond the HER2-positive subtype. To this end, we quantitatively assessed the wiring of signaling events in the individual subtypes to unravel the characteristics of HER-signaling. Our data along with a model-based analysis suggest that major parts of the intracellular signal transduction network are unchanged between the different breast cancer subtypes and that the clinical differences mostly come from the different levels at which these receptors are present in tumor cells as well as from the particular mutations that are present in individual tumors.Targeted therapies have shown striking success in the treatment of cancer over the last years. However, their specific effects on an individual tumor appear to be varying and difficult to predict. Using an integrative modeling approach that combines mechanistic and regression modeling, we gained insights into the response mechanisms of breast cancer cells due to different ligand–drug combinations. The multi-pathway model, capturing ERBB receptor signaling as well as downstream MAPK and PI3K pathways was calibrated on time-resolved data of the luminal breast cancer cell lines MCF7 and T47D across an array of four ligands and five drugs. The same model was then successfully applied to triple negative and HER2-positive breast cancer cell lines, requiring adjustments mostly for the respective receptor compositions within these cell lines. The additional relevance of cell-line-specific mutations in the MAPK and PI3K pathway components was identified via L1 regularization, where the impact of these mutations on pathway activation was uncovered. Finally, we predicted and experimentally validated the proliferation response of cells to drug co-treatments. We developed a unified mathematical model that can describe the ERBB receptor and downstream signaling in response to therapeutic drugs targeting this clinically relevant signaling network in cell line that represent three major subtypes of breast cancer. Our data and model suggest that alterations in this network could render anti-HER therapies relevant beyond the HER2-positive subtype.

TET1 Isoforms Have Distinct Expression Pattern, Localization and Regulation in Breast Cancer.

TET1 regulates gene expression by demethylating their regulatory sequences through the conversion of 5-methylcytosine to 5-hyroxymethylcytosine. TET1 plays important roles in tissue homeostasis. In breast cancer, TET1 was shown to play controversial roles. Moreover, TET1 has at least two isoforms (long and short) that have distinct expression pattern and apparently different functions in tissue development and disease including breast cancer. We hypothesized that TET1 isoforms have different expression patterns, localization and regulation in different types of breast cancer. To prove our hypothesis, we studied the expression of TET1 isoforms in basal and luminal breast cancer cell lines, as well as in basal and luminal breast cancer animal models. We also studied the effect of different hormones on the expression of the two isoforms. Moreover, we assessed the distribution of the isoforms between the cytoplasm and nucleus. Finally, we overexpressed the full length in a breast cancer cell line and tested its effect on cancer cell behavior. In this study, we demonstrate that while Estrogen and GnRH downregulate the expression of long TET1, they lead to upregulation of short TET1 expression. In addition, we uncovered that luminal cells show higher expression level of the long isoform. We also show that while all TET1 isoforms are almost depleted in a basal breast cancer animal model, the expression of the short isoform is induced in luminal breast cancer model. The short form is expressed mainly in the cytoplasm while the long isoform is expressed mainly in the nucleus. Finally, we show that long TET1 overexpression suppresses cell oncogenic phenotypes. In conclusion, our data suggest that TET1 isoforms have distinct expression pattern, localization and regulation in breast cancer and that long TET1 suppresses oncogenic phenotypes, and that further studies are necessary to elucidate the functional roles of different TET1 isoforms in breast cancer.

AXL/PLCγ1/PKCα as a Central Axis for PD‐L1 Regulation in Triple‐Negative Breast Cancer

Breast cancer is the most prevalent malignancy in women globally, accounting for 12% of all new annual cancer cases worldwide. Triple-negative breast cancer (TNBC), which is ER, PR and HER2 negative, represents 10-15% of breast cancers and displays an aggressive metastatic phenotype, with limited treatment options and poor patient prognosis. TNBC is associated with high expression levels of immune checkpoint programmed-death ligand 1 (PD-L1) compared with other breast cancer subtypes, and it is therefore amenable to immune checkpoint therapy. In this study, we analyzed the role of diacylglycerol (DAG)-regulated protein kinase C alpha (PKCα) in the control of PD-L1 expression in TNBC. Dataset analysis of 50 breast cancer cell lines revealed a prominent up-regulation of PKCα in TNBC, particularly in TNBC-B subtype, with a concomitant down-regulation of protein kinase C delta (PKCδ). This finding was authenticated in the TCGA-BRCA database. A significant correlation was found between PKCα expression and EMT markers, i.e., up-regulation of vimentin, Zeb1, TWIST1, SNAI2 and AXL, and E-cadherin down-regulation. High PKCα expression was also observed in TNBC patient-derived xenografts (PDXs). Immunofluorescence analysis revealed significant PKCα endogenous levels in the TNBC B cell line BT549, with strong "peripheral staining", a hallmark of its activated state. Conversely, a tenuous PKCα staining was detected in luminal breast cancer cells. Notably, TNBC-B cell lines express high levels of phosphorylated (active)-phospholipase C gamma 1 (PLCγ1), an enzyme responsible for DAG generation and PKCα activation. We also found a significant positive correlation between PKCα and PD-L1 expression in breast cancer cell lines. Notably, both RNAi silencing and pharmacological inhibition of PKCα or PLCγ1 reduced PD-L1 mRNA and protein levels in TNBC cells. We also show that AXL and PKCα mutually and positively control their expression in addition to up-regulating PD-L1, and they share a common gene expression signature that is highly enriched in cytokine, extracellular matrix, and motility pathways. Mechanistic analysis showed that inhibition of AXL, PLCγ1 or PKCα significantly reduced PD-L1 promoter activity in TNBC cells, suggesting a transcriptional control of the PDL1 (CD274) gene by this pathway. In summary, our results indicate that aberrant overactivation of AXL/PLCγ1/PKCα represents a cancer cell intrinsic mechanism for PD-L1 up-regulation in TNBC, suggesting a potential role for this axis in the control of tumor immunosurveillance. PKCα and AXL may be promising candidates for integrated targeted therapy in TNBC treatment.

CD68, CD86 and CD163 Expression Profile in Breast Cancer Molecular Subtypes

Background and aimBreast cancer (BC) is the second most common global cause of cancer deaths among women. Several immune cells are identified in the tumor microenvironment of BC patients, including tumor‐associated macrophages. We aimed at exploring the expression of distinct functional phenotypes using macrophages’ markers, where CD68 is a pan‐macrophage marker, CD86 is a marker expressed in polarized M1 subtype, and CD163 is expressed in M2 polarized subtype.MethodsA retrospective study was performed using 90 formalin‐fixed paraffin‐embedded BC specimens for the immunohistochemical analysis of CD68, CD86 and CD163. Also, an in silico tool, UALCAN, was used on a larger cohort (n=1,081) of BC patients to investigate the expression of these markers. The macrophages’ markers were then associated with the clinicopathological parameters of BC patients. Triple‐negative BC cell line “CAL‐51” and luminal BC cell line “MCF‐7” were used to collect their respective supernatants that were added to THP‐1 derived macrophages. Then CD86 and CD163 were assessed using western blot.ResultsThe pan‐marker of macrophages, CD68, along with the M1 CD86 marker and M2 CD163 marker, showed positive results in all the 90 investigated patients (Figure 1). CD86 expression was significantly associated with body mass index (BMI) and Ki‐67 proliferation marker. On the other hand, CD163 was significantly correlated with tumor size, estrogen, progesterone receptors, and BC molecular subtypes. Moreover, the high expression of CD86 and CD163 showed unfavorable outcome and survival of BC patients. In silicoanalysis revealed a significant increase in the CD86 expression in BC patients, especially in the triple‐negative subtype. Similarly, CD163 expression was found to be higher in the triple‐negative subgroup compared to the luminal group (Figure 2A). Additionally, in vitro work showed that the macrophages induced from the monocytic THP‐1 cell line express high levels of CD163 upon the exposure to conditioned media collected from the luminal BC cell line “MCF‐7”, thus showing M2 phenotype. On the contrary, CD163 expression was reduced upon the exposure to the conditioned media collected from the triple‐negative BC cell line “CAL51”, indicating more M1 phenotype (Figure 2B).ConclusionTumor‐associated macrophages are associated with cancer progression and molecular subtypes. Hence, identifying the macrophages polarization profiles in each molecular subtype might aid their use as potential therapeutic targets.

Clinically relevant CHK1 inhibitors abrogate wild-type and Y537S mutant ER\u03b1 expression and proliferation in luminal primary and metastatic breast cancer cells

BackgroundChallenges exist in the clinical treatment of luminal estrogen receptor α (ERα)-positive breast cancers (BCs) both to prevent resistance to endocrine therapy (ET) and to treat ET-resistant metastatic BCs (MBC). Therefore, we evaluated if kinases could be new targets for the treatment of luminal primary and MBCs.Methods ~ 170 kinase inhibitors were applied to MCF-7 cells either with adaptative or genetic resistance to ET drugs and both ERα levels and cell proliferation were measured. Robust-Z-score calculation identified AZD7762 (CHK1/CHK2 inhibitor) as a positive hit. Subsequently, Kaplan–Meier analyses of CHK1 and CHK2 impact on ERα-positive BC patients relapse-free-survival (RFS), bioinformatic evaluations of CHK1 and CHK2 expression and activation status as a function of ERα activation status as well as drug sensitivity studies in ERα-positive BC cell lines, validation of the impact of the ATR:CHK1 and ATM:CHK2 pathways on the control of ERα stability and BC cell proliferation via inhibitor- and siRNA-based approaches, identification of the molecular mechanism required for inhibitor-dependent ERα degradation in BC and the impact of CHK1 and CHK2 inhibition on the 17β-estradiol (E2):ERα signaling, synergy proliferation studies between ET-drugs and clinically relevant CHK1 inhibitors in different luminal BC cell lines, were performed.ResultsA reduced CHK1 expression correlates with a longer RFS in women with ERα-positive BCs. Interestingly, women carrying luminal A BC display an extended RFS when expressing low CHK1 levels. Accordingly, CHK1 and ERα activations are correlated in ERα-positive BC cell lines, and the ATR:CHK1 pathway controls ERα stability and cell proliferation in luminal A BC cells. Mechanistically, the generation of DNA replication stress rather than DNA damage induced by ATR:CHK1 pathway inhibition is a prerequisite for ERα degradation. Furthermore, CHK1 inhibition interferes with E2:ERα signaling to cell proliferation, and drugs approved for clinical treatment of primary and MBC (4OH-tamoxifen and the CDK4/CDK6 inhibitors abemaciclib and palbociclib) exert synergic effects with the CHK1 inhibitors in clinical trials for the treatment of solid tumors (AZD7762, MK8776, prexasertib) in preventing the proliferation of cells modeling primary and MBC.ConclusionsCHK1 could be considered as an appealing novel pharmacological target for the treatment of luminal primary and MBCs.

Protein kinase C targeting of luminal (T-47D), luminal/HER2-positive (BT474), and triple negative (HCC1806) breast cancer cells in-vitro with AEB071 (Sotrastaurin) is efficient but mediated by subtype specific molecular effects

PurposeProtein kinase C (PKC) plays a pivotal role in malignant cell proliferation, apoptosis, invasiveness and migration. However, its exploitation as therapeutic target in breast cancer has been merely explored. Here were evaluated the AEB071 (Sotrastaurin™) treatment efficiency of breast cancer cell lines derived from estrogen receptor positive (T-47D), estrogen/HER2 receptor positive (BT474), and triple negative (HCC1806) breast cancer cells under 2D (monolayer) and 3D (multicellular tumor spheroids) culture conditions. Additionally, spheroid cocultures of BC and N1 fibroblasts were analyzed.MethodsWe quantitatively assessed the proliferation capacity of breast cancer cells and fibroblasts as a function of AEB071 treatment using flow cytometry. The activities of PKC isoforms, substrates, and key molecules of the PKC signaling known to be involved in the regulation of tumor cell proliferation and cellular survival were additionally evaluated. Moreover, a multigene expression analysis (PanCancer Pathways assay) using the nanoString™ technology was applied.ResultsAll breast cancer cell lines subjected to this study were sensitive to AEB071 treatment, whereby cell proliferation in 2D culture was considerably (BT474) or moderately (HCC1806) retarded in G0/G1 or in G2/M phase (T-47D) of the cell cycle. Regardless of the breast cancer subtype the efficiency of AEB071 treatment was significantly lower in the presence of N1 fibroblast cells. Subtype specific driver molecules, namely IL19, c-myb, and NGFR were mostly affected by the AEB071 treatment.ConclusionA combined targeting of PKC and a subtype specific driver molecule might complement specified breast cancer treatment.

Abstract P3-09-12: Breast cancer derived GATA3 mutations disrupt luminal transcriptional network

Abstract GATA3 has been identified as one of the most frequently mutated genes in breast cancer. In the METABRIC cohort, among 1,980 patient cases, 230 breast cancers harbored GATA3 mutations (~12%). 75% of the GATA mutations were observed in luminal breast tumors (47% in luminal A, 28% in luminal B). The recent genomic data in metastatic breast cancer also showed that the frequency of GATA3 somatic mutations was even higher in the metastatic breast cancer cohort. Lung, lymph nodes, and brain metastases were observed in the GATA3 mutant breast cancer patients. Based on these patient genomic data, GATA3 mutations have been considered as cancer drivers, yet the functional consequences of GATA3 mutations are underexplored. We and other groups previously identified that patients carrying GATA3 mutations have diverse clinical features. More than 70% of cases are small nucleotide deletions or insertions (indel), while less than 30% are missense mutations. By classifying the GATA3 indel mutations into 4 groups, we observed distinct clinical features. Somatic mutations found in the GATA3 second zinc-finger domain (ZnFn2) are significantly associated with poorer patient outcomes including worse patient survival. ZnFn2 mutations are predominantly found in luminal B breast tumors, while splice site mutations are frequently found in luminal A breast tumors and associated with better patient survival. These distinct clinical features clearly suggest the differential impacts of GATA3 mutations on breast cancer cells. To dissect the function of GATA3 mutants, we developed a GATA3 mutant cell line (R330fs/WT T47D cells) by CRISPR, which endogenously expresses a heterozygous R330 frame-shift (R330fs) mutant. R330fs mutations are found in multiple data cohorts. The majority (>90%) of GATA3 mutations are heterozygous. Therefore, this R330fs mutant T47D cell line mimics the type of alteration found in patients. Importantly, we found that the R330fs mutation induces transcriptional reprogramming in T47D luminal breast cancer cells leading to more aggressive phenotypes such as faster tumor growth. In the mutant cells, many epithelial marker genes (such as progesterone receptor and TFF1) were down-regulated while mesenchymal marker genes (such as TWIST1 and SNAI2) are up-regulated suggesting that R330fs induces a partial EMT in T47D cells. We also found that transcriptional changes in the R330fs mutant cells were strongly associated with redistribution of GATA3, Estrogen Receptor alpha, and FOXA1. Gene expression profiles in the other GATA3 mutant breast cancer cell lines also suggest similar genomic alterations. These results suggest the active roles of GATA3 mutations during breast cancer development. Citation Format: Motoki Takaku, Mika Saotome, Renju Nair. Breast cancer derived GATA3 mutations disrupt luminal transcriptional network [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-12.

Mass cytometric and transcriptomic profiling of epithelial-mesenchymal transitions in human mammary cell lines

Epithelial-mesenchymal transition (EMT) equips breast cancer cells for metastasis and treatment resistance. However, detection, inhibition, and elimination of EMT-undergoing cells is challenging due to the intrinsic heterogeneity of cancer cells and the phenotypic diversity of EMT programs. We comprehensively profiled EMT transition phenotypes in four non-cancerous human mammary epithelial cell lines using a flow cytometry surface marker screen, RNA sequencing, and mass cytometry. EMT was induced in the HMLE and MCF10A cell lines and in the HMLE-Twist-ER and HMLE-Snail-ER cell lines by prolonged exposure to TGFβ1 or 4-hydroxytamoxifen, respectively. Each cell line exhibited a spectrum of EMT transition phenotypes, which we compared to the steady-state phenotypes of fifteen luminal, HER2-positive, and basal breast cancer cell lines. Our data provide multiparametric insights at single-cell level into the phenotypic diversity of EMT at different time points and in four human cellular models. These insights are valuable to better understand the complexity of EMT, to compare EMT transitions between the cellular models used here, and for the design of EMT time course experiments.

GATA3 Truncation Mutants Alter EMT Related Gene Expression via Partial Motif Recognition in Luminal Breast Cancer Cells.

GATA3 is known to be one of the most frequently mutated genes in breast cancer. More than 10% of breast tumors carry mutations in this gene. However, the functional consequence of GATA3 mutations is still largely unknown. Clinical data suggest that different types of GATA3 mutations may have distinct roles in breast cancer characterization. In this study, we have established three luminal breast cancer cell lines that stably express different truncation mutants (X308 splice site deletion, C321 frameshift, and A333 frameshift mutants) found in breast cancer patients. Transcriptome analysis identified common and distinct gene expression patterns in these GATA3 mutant cell lines. In particular, the impacts on epithelial-to-mesenchymal transition (EMT) related genes are similar across these mutant cell lines. Chromatin localization of the mutants is highly overlapped and exhibits non-canonical motif enrichment. Interestingly, the A333 frameshift mutant expressed cells displayed the most significant impact on the GATA3 binding compared to X308 splice site deletion and C321fs mutants expressed cells. Our results suggest the common and different roles of GATA3 truncation mutations during luminal breast cancer development.

Biglycan Promotes Cancer Stem Cell Properties, NFκB Signaling and Metastatic Potential in Breast Cancer Cells.

Simple SummaryBreast cancer stem cells (BCSCs) are a small sub-population of cells within tumors with high metastatic potential. We identified biglycan (BGN) as a prospective molecular target in BCSCs that regulates the aggressive phenotypes of these cells. These findings establish a foundation for the development of therapeutics against BGN to eliminate BCSCs and prevent metastatic breast cancer.It is a major challenge to treat metastasis due to the presence of heterogenous BCSCs. Therefore, it is important to identify new molecular targets and their underlying molecular mechanisms in various BCSCs to improve treatment of breast cancer metastasis. Here, we performed RNA sequencing on two distinct co-existing BCSC populations, ALDH+ and CD29hi CD61+ from PyMT mammary tumor cells and detected upregulation of biglycan (BGN) in these BCSCs. Genetic depletion of BGN reduced BCSC proportions and tumorsphere formation. Furthermore, BCSC associated aggressive traits such as migration and invasion were significantly reduced by depletion of BGN. Glycolytic and mitochondrial metabolic assays also revealed that BCSCs exhibited decreased metabolism upon loss of BGN. BCSCs showed decreased activation of the NFκB transcription factor, p65, and phospho-IκB levels upon BGN ablation, indicating regulation of NFκB pathway by BGN. To further support our data, we also characterized CD24−/CD44+ BCSCs from human luminal MCF-7 breast cancer cells. These CD24−/CD44+ BCSCs similarly exhibited reduced tumorigenic phenotypes, metabolism and attenuation of NFκB pathway after knockdown of BGN. Finally, loss of BGN in ALDH+ and CD29hi CD61+ BCSCs showed decreased metastatic potential, suggesting BGN serves as an important therapeutic target in BCSCs for treating metastasis of breast cancer.

The combination of TRAIL and the Smac mimetic LCL-161 induces an irreversible phenotypic change of MCF-7 breast cancer cells

IntroductionBreast cancer is the most common malignancy affecting women. Although the prognosis generally is good, a substantial number of patients still suffer from relapse, emphasizing the need for novel treatments. Smac mimetics were developed to facilitate cell death by blocking inhibitor of apoptosis proteins (IAPs). It has been suggested that TNF-related apoptosis inducing ligand (TRAIL) can be used together with Smac mimetics to induce cancer cell death. MethodsCell viability was studied with Trypan blue staining and Annexin V assay, siRNA was used to downregulate specific proteins, protein levels were estimated with Western blot, and mRNA levels were analyzed with qPCR, microarray and RNA-seq. For global expression, groups were compared with principal component analysis and the limma package in R. Gene enrichment was analyzed with Fisher's test. For other experiments, significance of difference was tested by one-way ANOVA, followed by Tukey's HSD test. ResultsThe combination of Smac mimetic LCL-161 and TRAIL induces an irreversible change in phenotype, but not cell death, of luminal MCF-7 breast cancer cells. The cells become small and circular and dissociate from each other and the effect could not be reversed by returning the cells to regular growth medium. The morphology change could be prevented by caspase inhibition using z-VAD-FMK and downregulation of caspase-8. Caspase-7 is also indicated to be of importance since downregulation of this caspase resulted in fewer morphologically changed cells. Enrichment analyses of changes in global gene expression demonstrated that genes associated with estrogen receptor (ER) signaling are downregulated, whereas nuclear factor kappa B- (NF-κB) and interferon- (IFN) driven genes are upregulated in altered cells. However, inhibition of these pathways did not influence the change in morphology. Induction of IFN-induced genes were potentiated but NF-ĸB-driven genes were slightly suppressed by caspase inhibition. ConclusionsThe results demonstrate that LCL-161 and TRAIL can irreversibly alter the MCF-7 breast cancer cell phenotype. However, the changes in morphology and global gene expression are mediated via separate pathways.

Dermatan Sulfate Affects Breast Cancer Cell Function via the Induction of Necroptosis.

Dermatan sulfate (DS) is widespread in the extracellular matrix (ECM) of animal tissues. This glycosaminoglycan is characterized by a variable structure, which is reflected in the heterogeneity of its sulfation pattern. The sulfate groups are responsible for the binding properties of DS, which determine an interaction profile of this glycan. However, the detailed role of DS in biological processes such as the neoplasm is still poorly understood. The aim of the study was to assess the effects of the structural variants of DS on breast cancer cells. We found that DS isoforms from normal and fibrotic fascia as well as from intestinal mucosa were able to quickly induce oxidative stress in the cytoplasm and affect the mitochondrial function in luminal breast cancer cells. Moreover, the variants caused the necroptosis of the cells most likely via the first of these mechanisms. This death was responsible for a reduction in the viability and number of breast cancer cells. However, the dynamics and intensity of all of the DS variants-triggered effects were strongly dependent on the cell type and the structure of these molecules. The most pronounced activity was demonstrated by those variants that shared structural features with the DS from the tumor niche.

Lineage-specific silencing of PSAT1 induces serine auxotrophy and sensitivity to dietary serine starvation in luminal breast tumors.

SUMMARYA major challenge of targeting metabolism for cancer therapy is pathway redundancy, in which multiple sources of critical nutrients can limit the effectiveness of some metabolism-targeted therapies. Here, we analyze lineage-dependent gene expression in human breast tumors to identify differences in metabolic gene expression that may limit pathway redundancy and create therapeutic vulnerabilities. We find that the serine synthesis pathway gene PSAT1 is the most depleted metabolic gene in luminal breast tumors relative to basal tumors. Low PSAT1 prevents de novo serine biosynthesis and sensitizes luminal breast cancer cells to serine and glycine starvation in vitro and in vivo. This PSAT1 expression disparity preexists in the putative cells of origin of basal and luminal tumors and is due to luminal-specific hypermethylation of the PSAT1 gene. Our data demonstrate that luminal breast tumors are auxotrophic for serine and may be uniquely sensitive to therapies targeting serine availability.

Verification of electron beam parameters in an intraoperative linear accelerator using dosimetric and radiobiological response methods.

BackgroundThe availability of linear accelerators (linac) for research purposes is often limited and therefore alternative radiation sources are needed to conduct radiobiological research. The National Centre for Radiation Research in Poland recently developed an intraoperative mobile linac that enables electron irradiation at energies ranging from 4 to 12 MeV and dose rates of 5 or 10 Gy/min. The present study was conducted to evaluate the electron beam parameters of this intraoperative linac and to verify the set-up to evaluate out-of-field doses in a water phantom, which were determined through dosimetric and biological response measurements.Materials and methodsThe distribution of radiation doses along and across the radiation beam were measured in a water phantom using a semiconductor detector and absolute doses using an ionisation chamber. Two luminal breast cancer cell lines (T-47D and HER2 positive SK-BR-3) were placed in the phantom to study radiation response at doses ranging from 2 to 10 Gy. Cell response was measured by clonogenic assays.Results and ConclusionThe electron beam properties, including depth doses and profiles, were within expected range for the stated energies. These results confirm the viability of this device and set-up as a source of megavoltage electrons to evaluate the radiobiological response of tumour cells.