Basal Breast Cancer Cell Lines Research Articles

KRT14 as a potential prognostic marker for metastasis in metaplastic breast carcinoma

Metaplastic breast cancer (MBC) is an aggressive breast cancer subtype with poor prognosis. To elucidate underlying genomic aberrations driving MBC, we conducted integrated transcriptomic analyses of MBC tumors and basal breast cancer cell lines with various transitioning phases (xE = epithelial phase clone, xM = mesenchymal phase clone and xEM = Epithelial-mesenchymal clone). Differential gene expression analysis between MBC vs non-MBC samples as well as between epithelial-mesenchymal (xEM) transitioning cells vs parental cells revealed 12 commonly dysregulated genes intersected from both the datasets meditates pathways governing EMT, migration, proliferation and metastasis. Further investigation onto those genes based on multi-omics approaches that include survival analysis, correlated expression patterns and interactomics indicated KRT14 had a key role in promoting MBC aggression through Epithelial mesenchymal transition leading to metastasis. Moreover, NPR3 was found to be a possible link between KRT14 and EMT in metastasis. To our knowledge, this is the first evident gene expression study to investigate the potential association between the expression of KRT14 and NPR3 and metastasis in MBC, and their potential involvement in the epithelial-mesenchymal transition (EMT) process. These mechanistic and prognostic insights gained can lead to targeted therapeutics against this rare, refractory disease. Overall, this work highlights the effectiveness of integrated multi-omics in revealing gene expression pattern and predictive biomarkers for metaplastic breast cancer subtypes with poor prognosis.

Abstract A023: Sensitivity/resistance mechanisms to KDM5 inhibition in basal breast cancer

Abstract We previously described that the KDM5B histone H3 lysine 4 demethylase is amplified and overexpressed in luminal estrogen receptor positive breast cancer, and its activity is associated with resistance to endocrine therapies. We have found that the KDM5A paralog is specifically amplified and overexpressed in basal breast cancer, providing a novel therapeutic opportunity for an aggressive breast cancer subtype. To identify modulators of KDM5 inhibitor sensitivity in KDM5A-amplified basal breast cancer, we performed a genome wide CRISPR viability screen in a KDM5A-amplified basal breast cancer cell line. Our CRISPR screen results and follow up studies implicate Rho-GTPase signaling and the transcriptional repressor ZBTB7A as modulators of cellular response to KDM5 inhibition. We are currently investigating the underlying mechanisms for how these components alter sensitivity/resistance to KDM5 inhibition. Our results suggest potential therapeutic opportunities of inhibiting the KDM5 family of H3K4 demethylases in basal breast cancer. Citation Format: Benedetto DiCiaccio, Evangelia Papachristou, Malvina Papanastasiou, Andrew Reiter, Jason Carroll, Kornelia Polyak. Sensitivity/resistance mechanisms to KDM5 inhibition in basal breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A023.

Abstract A018: KDM4C histone demethylase connects redox balance to chromatin remodeling via histone H3 tail clipping

Abstract Triple-negative breast cancer (TNBC) is a subtype accounting for 20-30% all breast cancer cases and is a highly aggressive disease with inferior prognosis. The acquired resistance is a major obstacle for efficient therapy and discovery of novel clinically-actionable targets has become an urgent need. In this study, we carried out an in-depth functional characterization of histone demethylase KDM4C in TNBC and uncovered a novel mechanism underlying KDM4C-driven tumorigenesis. KDM4C is the second most frequently amplified histone demethylase in TNBC. Genetic knockdown or pharmacological inhibition of KDM4C in two amplified basal breast cancer cell lines, SUM149 and HCC1954, drastically suppressed primary tumor growth in vitro and in vivo. Transcriptomic analysis underlined oxidative phosphorylation impairment as functional consequences of KDM4C blockade. Surprisingly, integrating series of histone ChIP-seq and ATAC-seq showed that KDM4C suppression caused accessible chromatin remodeling without substantial changes of its canonical substrates H3K9me3 and H3K36me3. Rather KDM4C loss caused proteolytic cleavage at histone 3 N-terminus (Ala21 site) identified by histone mass spectrometry. Protease inhibitor array pointed out cathepsin L (CTSL) as the endopeptidase mediating this procedure. KDM4C blockade induced CTSL activation and promoted H3 tail clipping at around 30% CTSL binding sites, which was associated with restricted chromatin accessibility and transcriptomic reprogramming. Proteomic interactome profiling revealed that grainyhead like transcription factor 2 (GRHL2) tightly binds to CTSL. In addition, GRHL2 knockout induced nearly complete loss of CTSL chromatin binding, demonstrating its role as a recruiter of CTSL to the chromatin. KDM4C serves as either a direct or indirect suppressor for CTSL-mediated H3 cleavage. The latter one was largely mediated by redox imbalance. Metabolomic profiling showed that KDM4C inhibition strongly dampened intracellular glutathione (GSH) levels and hence produced more reactive oxygen species (ROS) and alleviated mitochondrial respiration. This procedure was grounded on the mechanism that KDM4C blockade decreased expression of GSH synthesis rate-limiting enzyme glutamate-cysteine ligase catalytic subunit (GCLC) via CTSL-mediated H3 tail clipping. Knockout of CTSL rescued KDM4C blockade-associated metabolic dysfunction, confirming KDM4C links redox homeostasis and chromatin remodeling. Finally, we found KDM4C-CTSL-GSH axis was associated with cisplatin resistance in TNBC patients. Co-targeting KDM4C and GSH production efficiently improved cisplatin response in TNBC cells. Our study provided detailed and unbiased evidence supporting a novel non-canonical role of KDM4C in preventing CTSL-mediated histone clipping and maintaining redox balance, opening up possibilities to improve chemotherapy through targeting this circus. Citation Format: Zheqi Li, Guillermo Peluffo, Laura E. Stevens, Xintao Qiu, Shawn B. Egri, Malvina Papanastasiou, Natalie Kingston, Clive S. D'Santos, Eva Papachristou, Kyle Evans, Ji-Heui Seo, Kendell Clement, Daniel Temko, Muhammad Ekram, Anton Simeonov, Stephen C. Kales, Ganesha Rai, Madhu Lal-Nag, David J. Maloney, Ajit Jadhav, Franziska Michor, Alex Meissner, Jason S. Carroll, Matthew L. Freedman, Henry W. Long, Jacob D. Jaffe, Kornelia Polyak. KDM4C histone demethylase connects redox balance to chromatin remodeling via histone H3 tail clipping. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A018.

AKT Regulation of ORAI1-Mediated Calcium Influx in Breast Cancer Cells.

Simple SummaryA remodeling in calcium homeostasis and the protein kinase AKT signaling pathway often promotes tumorigenic traits in cancer cells. Changes in calcium signaling can be mediated through altered expression or activity of calcium channels and pumps, which constitute a class of targetable therapeutic targets. Currently, the interplay between the two signaling pathways in breast cancer cells is unclear. A better understanding of the association between calcium and AKT signaling, and the molecular players involved may identify novel therapeutic strategies for breast cancers with abnormal AKT signaling. Using fluorescence calcium imaging and gene silencing/knockout techniques, we showed that increased AKT activation results in increased calcium entry, and that this is mediated through ORAI1 calcium channels. Future studies exploring therapeutic strategies to target PTEN-deficient or hyperactivated AKT cancers should consider this novel correlation between AKT activation and ORAI1-mediated calcium influx.Although breast cancer cells often exhibit both abnormal AKT signaling and calcium signaling, the association between these two pathways is unclear. Using a combination of pharmacological tools, siRNA and CRISPR/Cas9 gene silencing techniques, we investigated the association between PTEN, AKT phosphorylation and calcium signaling in a basal breast cancer cell line. We found that siRNA-mediated PTEN silencing promotes AKT phosphorylation and calcium influx in MDA-MB-231 cells. This increase in AKT phosphorylation and calcium influx was phenocopied by the pharmacological AKT activator, SC79. The increased calcium influx associated with SC79 is inhibited by silencing AKT2, but not AKT1. This increase in calcium influx is suppressed when the store-operated calcium channel, ORAI1 is silenced. The results from this study open a novel avenue for therapeutic targeting of cancer cells with increased AKT activation. Given the association between ORAI1 and breast cancer, ORAI1 is a possible therapeutic target in cancers with abnormal AKT signaling.

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.

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.

Comprehensive landscape of epigenetic-dysregulated lncRNAs reveals a profound role of enhancers in carcinogenesis in BC subtypes

Aberrant expression of long non-coding RNAs (lncRNA) is associated with altered DNA methylation and histone modifications during carcinogenesis. However, identifying epigenetically dysregulated lncRNAs and characterizing their functional mechanisms in different cancer subtypes are still major challenges for cancer studies. In this study, we systematically analyzed the epigenetic alterations of lncRNAs at important regulatory elements in three breast cancer subtypes. We identified 87, 691, and 1,197 epigenetically dysregulated lncRNAs in luminal, basal, and claudin-low subtypes of breast cancer, respectively. The landscape of epigenetically dysregulated lncRNAs at enhancer elements revealed that epigenetic changes of the majority of lncRNAs occurred in a subtype-specific manner and contributed to subtype-specific biological functions. We identified six acetylation of lysine 27 on histone H3 (H3K27ac)-dysregulated lncRNAs and three DNA methylation-dysregulated lncRNAs (CTC-303L1.2, RP11-738B7.1, and SLC26A4-AS1) as prognostic biomarkers of basal subtype. These lncRNAs were involved in immune response-related biological functions. Treatment of the basal breast cancer cell line MDA-MB-468 with CREBBP/EP300 bromodomain inhibitors downregulated H3K27 acetylation levels and caused a decrease in the expression of five H3K27ac-dysregulated lncRNAs (LINC00393, KB-1836B5.1, RP1-140K8.5, AC005162.1, and AC020916.2) and inhibition of the growth of breast cancer cells. One epigenetically dysregulated lncRNA (LINC01983) and four lncRNA regulators (UCA1, RP11-221J22.2, RP11-221J22.1, and RP1-212P9.3) were identified as prognostic biomarkers of the luminal molecular subtype of breast cancer by controlling the tumor necrosis factor (TNF) signaling pathway, T helper (Th)17 cell differentiation, and T cell migration. Finally, our results highlighted a profound role of enhancer-related H3K27ac-dysregulated lncRNAs, DNA methylation-dysregulated lncRNAs, and lncRNA regulators in breast cancer subtype carcinogenesis and their potential prognostic value.

Abstract 1424: Her3 up regulation in response to AKT and PI3K inhibition causes differential responses dependent on the presence of neuregulin isoforms in the microenvironmental milieu

Abstract Introduction: Triple Negative Breast Cancer (TNBC) is an aggressive form of Breast Cancer (BC) affecting ~20% of BC patients. TNBC is a highly heterogenous disease and numerous tyrosine kinase inhibitors (TKI) targeting different pathways including the PI3K, AKT and MAPK pathway have shown limited benefit in the clinical setting. Intrinsic and extrinsic resistance mechanisms to TKI are the main reason of therapeutic failure. Signalling from the human epidermal growth factor receptor (HER) family of proteins is dependent on a well-orchestrated series of interactions between family members to form either homo- or heterodimers. The heterodimeric complex formed with HER3 is a particularly potent oncogenic signalling unit that can act as a driver of cancer growth. HER3 upregulation in response to small molecule inhibitors has been shown to rescue a large number of cancers including TNBC. In this study we aim to characterize the extent of HER3 reliance in TNBC and to define the effect of neuregulin isoforms in TNBC cancers. Method and Materials: Basal and Claudin type Breast cancer cell lines were treated with a range of small molecule inhibitors, known to exhibit limited efficacy as single agents, including the AKT inhibitor (GDC0068), the PI3K inhibitor (GDC0077) and the EGFR inhibitors (Gefitinib and Lapatinib) or a combination of these drugs in the presence or absence of the HER3 ligand neuregulin. The effect these inhibitors on Akt/ MAPK signalling was characterized using specific biomarkers of the respective pathways. Combination therapy was evaluated in human cancer cell lines through Annexin V staining. Results: Basal (HCC-70 and MDA-MB-468) and Caudin type (MDA-MB-231) TNBC cell lines displayed differential reliance on the HER family of receptors. Dynamic HER3 upregulation was predominant in the TNBC cells that show HER signaling reliance coinciding with the basal subtype. Furthermore, the presence of the natural ligand neuregulin showed potent signaling through the HER3-AKT pathway and significantly diminished the efficacy of the inhibitors tested. We report that neuregulin augments the HER3 feedback mechanism for continued proliferation in TNBC. Interestingly, we show that neuregulin mediated resistance is dependent on the type and dose of the neuregulin isoform. Neuregulin 1β showed potent proliferative effects and overcoming this effect required a 10 fold increase in the drug concentration. In contrast, the presence of neuregulin 1α isoform may paradoxically confer sensitivity to small molecule inhibitors. We show that cellular proliferation is maintained through both the PI3K and MAPK pathway and single targeting is inefficient in overcoming resistance. We demonstrate that combination strategies that block compensatory mechanisms are effective strategies to neuregulin dependent and independent resistance mechanisms. Conclusion: The highly dynamic nature of HER3 expression and signalling combined with redundant HER signalling and downstream mechanisms allow for continued signalling in a subtype of TNBC patients. Targeted therapy to ensure complete abrogation of HER signalling is essential for effective therapeutic response. In conclusion, TNBC cancers display differential mechanism of resistance that should be adequately addressed for therapeutic strategies. Citation Format: Nicoleta Sinevici, Pedram Heidari, Umar Mahmood. Her3 up regulation in response to AKT and PI3K inhibition causes differential responses dependent on the presence of neuregulin isoforms in the microenvironmental milieu [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1424.

Polo-like kinase 1 (Plk1) inhibition synergizes with taxanes in triple negative breast cancer.

Within triple negative breast cancer, several molecular subtypes have been identified, underlying the heterogeneity of such an aggressive disease. The basal-like subtype is characterized by mutations in the TP53 gene, and is associated with a low pathologic complete response rate following neoadjuvant chemotherapy. In a genome-scale short hairpin RNA (shRNA) screen of breast cancer cells, polo-like kinase 1 (Plk1) was a frequent and strong hit in the basal breast cancer cell lines indicating its importance for growth and survival of these breast cancer cells. Plk1 regulates progression of cells through the G2-M phase of the cell cycle. We assessed the activity of two ATP-competitive Plk1 inhibitors, GSK461364 and onvansertib, alone and with a taxane in a set of triple negative breast cancer cell lines and in vivo. GSK461364 showed synergism with docetaxel in SUM149 (Combination Index 0.70) and SUM159 (CI, 0.62). GSK461364 in combination with docetaxel decreased the clonogenic potential (interaction test for SUM149 and SUM159, p<0.001 and p = 0.01, respectively) and the tumorsphere formation of SUM149 and SUM159 (interaction test, p = 0.01 and p< 0.001). In the SUM159 xenograft model, onvansertib plus paclitaxel significantly decreased tumor volume compared to single agent paclitaxel (p<0.0001). Inhibition of Plk1 in combination with taxanes shows promising results in a subset of triple negative breast cancer intrinsically resistant to chemotherapy. Onvansertib showed significant tumor volume shrinkage when combined with paclitaxel in vivo and should be considered in clinical trials for the treatment of triple negative cancers.

TFAP2C regulates carbonic anhydrase XII in human breast cancer

The expression of carbonic anhydrase XII (CA12) is associated with the expression of estrogen receptor alpha (ERα) in breast cancer and is linked to a good prognosis with a lower risk of metastasis. Transcription Factor Activator Protein 2γ (TFAP2C, AP-2γ) governs luminal breast cancer phenotype through direct and indirect regulation of ERα and ERα-associated genes, GATA3, FOXA1, EGFR, CDH1, DSP, KRT7, FBP1, MYB, RET, KRT8, MUC1, and ERBB2-genes which are responsible for the luminal signature in breast cancer. Herein, utilizing chromatin immunoprecipitation and direct sequencing (ChIP-seq), we show that CA12 is regulated by AP-2γ through binding with its promoter region in luminal breast cancer cell lines and indirectly through a distal estrogen-responsive region in ERα-positive cell lines by upregulation of ERα. CA12 is transcriptionally silenced in basal breast cancer cell lines through histone deacetylation and CpG methylation of the promoter region and can be re-activated with Trichostatin A (histone deacetylase inhibitor) and/or 5-aza-dC (an inhibitor of DNA methylation). Strong concordance in co-expression of CA12 and ESR1 (R2 = 0.1128, p = 0486) and TFAP2C (R2 = 0.1823, p = 0.0105) was found using a panel of primary breast tumor samples (n = 35), supporting a synergetic role of AP-2γ and ERα in activation of CA12. Our results highlight the essential role of AP-2γ in maintaining the luminal breast cancer phenotype and provide evidence that epigenetic mechanisms silence luminal gene expression in the basal phenotype. Additional studies to decipher mechanisms that drive epigenetic silencing of AP-2γ target genes are a critical area for further research.

Chromatin-informed inference of transcriptional programs in gynecologic and basal breast cancers

Chromatin accessibility data can elucidate the developmental origin of cancer cells and reveal the enhancer landscape of key oncogenic transcriptional regulators. We develop a computational strategy called PSIONIC (patient-specific inference of networks informed by chromatin) to combine chromatin accessibility data with large tumor expression data and model the effect of enhancers on transcriptional programs in multiple cancers. We generate a new ATAC-seq data profiling chromatin accessibility in gynecologic and basal breast cancer cell lines and apply PSIONIC to 723 patient and 96 cell line RNA-seq profiles from ovarian, uterine, and basal breast cancers. Our computational framework enables us to share information across tumors to learn patient-specific TF activities, revealing regulatory differences between and within tumor types. PSIONIC-predicted activity for MTF1 in cell line models correlates with sensitivity to MTF1 inhibition, showing the potential of our approach for personalized therapy. Many identified TFs are significantly associated with survival outcome. To validate PSIONIC-derived prognostic TFs, we perform immunohistochemical analyses in 31 uterine serous tumors for ETV6 and 45 basal breast tumors for MITF and confirm that the corresponding protein expression patterns are also significantly associated with prognosis.

Abstract 3370: Chromatin-informed inference of transcriptional programs in gynecologic and basal breast cancers

Abstract Epigenomic data on transcription factor occupancy and chromatin accessibility can elucidate the developmental origin of cancer cells and reveal the enhancer landscape of key oncogenic transcriptional regulators. We develop a computational strategy called PSIONIC (patient-specific inference of networks informed by chromatin) to combine cell line chromatin accessibility data with large tumor expression data sets and model the effect of enhancers on transcriptional programs in multiple cancers. We generated a new ATAC-seq data set profiling chromatin accessibility in gynecologic and basal breast cancer cell lines and applied PSIONIC to 723 patient and 96 cell line RNA-seq profiles from ovarian, uterine, and basal breast cancers. Our computational framework enables us to share information across tumors to learn patient-specific TF activities, revealing regulatory differences between and within tumor types. Many of theidentified TFs were significantly associated with survival outcome in basal breast, uterine serous and endometrioid carcinomas. To validate one PSIONIC-derived prognostic TF, we performed immunohistochemical analyses in 31 uterine serous tumors for ETV6 and confirmed that the corresponding protein expression pattern was also significantly associated with prognosis. Moreover, PSIONIC-predicted activity for MTF1 in cell line models correlated with sensitivity to MTF1 inhibition, showing the potential of our approach for personalized therapy. Citation Format: Hatice Ulku Osmanbeyoglu, Fumiko Shimizu, Angela Rynne-Vidal, Tsz-Lun Yeung, Petar Jelinic, Samuel C. Mok, Gabriela Chiosis, Douglas A. Levine, Christina Leslie. Chromatin-informed inference of transcriptional programs in gynecologic and basal breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3370.

Crumbs protein homolog 3 (CRB3) expression is associated with oestrogen and progesterone receptor positivity in breast cancer.

The crumbs protein homolog 3 (CRB3) regulates the tight junction to help maintain epithelial polarity. Altered CRB3 expression was associated with carcinogenesis of epithelial cells. This study detected CRB3 expression in 192 cases of breast cancer tissues and in the Molecular Taxonomy of Breast Cancer International Consortium (Metabric) and The Cancer Genome Atlas (TCGA) datasets for association with triple negative breast cancer (TNBC) phenotypes. The in vitro experiments confirm the ex vivo data. The data showed that levels of both CRB3 mRNA and protein were associated with TNBC phenotypes, ie, 41.1% (39/95) of ER+ breast cancer was CRB3‐positive, whereas 26.9% (25/93) ER– tumour was CRB3‐positive (P = 0.046). Moreover, 47.6% (30/63) of PR+ breast cancer was CRB3‐positive vs 28.4% (33/116) PR‐ tumours positive for CRB3 (P = 0.013). In addition, 40.1% (27/66) of ER+/PR+ tumour was CRB3‐positive, but only 22.4% (19/85) of TNBC showed CRB3 expression (P = 0.048). Indeed, levels of CRB3 mRNA were higher in non‐TNBC than TNBC in both Metabric (P = 3.682e‐10) and TCGA datasets (P = 2.501e‐07). The in vitro data showed that CRB3 expression was higher in luminal (MCF7 and T47D) than in HER2 (MDA‐MB‐453 and SK‐BR‐3) and basal (MDA‐MB‐231 and BT‐549) breast cancer cell lines. More interestingly, ERα regulated expression of CRB3 protein in MCF7 and BT‐549 cells and ERα expression was associated with CRB3 expression in breast cancer tissues specimens. This study demonstrated that ERα could be a novel regulator for CRB3 expression in breast cancer.

ORAI1 and ORAI3 in Breast Cancer Molecular Subtypes and the Identification of ORAI3 as a Hypoxia Sensitive Gene and a Regulator of Hypoxia Responses.

The remodeling of specific calcium-permeable ion channels is a feature of some breast cancer subtypes. ORAI1 is a protein that forms a calcium-permeable ion channel responsible for store-operated calcium entry (SOCE) in a variety of cell types. ORAI3, a related isoform, is not a regulator of SOCE in most cell types. However, ORAI3 does control SOCE in many estrogen receptor-positive breast cancer cell lines, where it also controls proliferation. ORAI1 is a well-characterized regulator of the proliferation and migration of many basal breast cancer cells; however, the role of ORAI3 in these types of breast cancer cells remains unclear. Here, we sought to define ORAI1 and ORAI3 expression in breast cancer cell lines of different molecular subtypes and assess the potential role and regulation of ORAI3 in basal breast cancer cells. Our study demonstrates that elevated ORAI1 is a feature of basal-like breast cancers, while elevated ORAI3 is a feature of luminal breast cancers. Intriguingly, we found that ORAI3 is over-expressed in the mesenchymal subtype of triple-negative breast cancer. Given this, we assessed ORAI3 levels in the presence of two inducers of the mesenchymal phenotype, hypoxia and epidermal growth factor (EGF). Hypoxia induced ORAI3 levels in basal breast cancer cell lines through a pathway involving hypoxia-inducible factor-1 alpha (HIF1α). The silencing of ORAI3 attenuated hypoxia-associated phosphorylation of the EGF receptor (EGFR) and the expression of genes associated with cell migration and inflammatory/immune responses in the MDA-MB-468 model of basal breast cancer. Although elevated ORAI3 levels were not associated with survival; basal, estrogen receptor-negative and triple-negative breast cancers with high ORAI3 and low ORAI1 levels were associated with poorer clinical outcomes. This study defines ORAI3 as a potential fine-tuner for processes relevant to the progression of basal breast cancers.

Abstract 5179: Uncovering the signaling landscape controlling breast cancer cell migration identifies splicing PRPF4B as a metastasis driver

Abstract Metastasis is the major cause of death in cancer patients and migration of cancer cells from the primary tumor to distant sites is the prerequisite of metastasis formation. Here we applied an imaging-based RNAi phenotypic cell migration screen uing two highly migratory basal breast cancer cell lines (Hs578T and MDA-MB-231 to provide a repository of candidate metastasis drug targets. We screened ~4,200 individual target genes covering most cell signaling components. We discovered 133 and 113 migratory modulators of Hs578T and MDA-MB-231, respectively, of which 45 genes were common denominators of cell migration. Interaction networks of candidate migratory modulators were in common with networks of different clinical breast cancer prognostic and metastasis classifiers. The splicing factors PRPF4B and BUD31 and the transcription factor BPTF were amplified in human primary breast tumors and the expression was associated with metastasis-free survival. Depletion of PRPF4B, BUD31 and BPTF caused primarily downregulation of genes involved in focal adhesion and ECM-interaction pathways. PRPF4B was essential for TNBC cell migration and critical for breast cancer metastasis formation in vivo, making PRPF4B a candidate for further drug development. Our systematic phenotypic screen provides an important repository for signaling determinants that functionally drive cancer cell migration. Citation Format: Bob van de Water, Erik Danen, Esmee Koedoot, Maria Rogkoti, Michiel Fokkelman, Sylvia Le Devedec, John Martens, Peter Stoilov, John Foekens. Uncovering the signaling landscape controlling breast cancer cell migration identifies splicing PRPF4B as a metastasis driver [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5179.

Abstract 4671: Rapid, no-wash measurement of immune checkpoint molecules expression induced by interaction with peripheral blood mononuclear cells in breast and cervical cancer cell models

Abstract The purpose of this study was to examine the pathways involved in the induction of immune checkpoint molecules in basal breast and cervical cancer cell lines by measuring classical biomarker expression using AlphaLISA no-wash homogeneous assays. Programmed cell death-ligand 1 (PD-L1) expression increases with tumor severity in basal-like breast cancer and is enhanced in intraepithelial neoplasia and cervical cancers. Basal-breast tumors can adapt to lymphocytic infiltration by responding to heightened concentrations of interferon gamma (IFN-y) secreted by Type 1 helper T cells with upregulation of PD-L1 protein allowing the tumors to evade immune targeting and reduce the immune response. We show here that human peripheral blood mononuclear cells (PBMCs) secrete IFN-y and other cytokines in response to stimulation with CD3/CD38 Dynabeads. We then examined modulation in expression of immune checkpoint molecules (PD-L1 and others) in HCC38 (basal breast cancer-derived) and HeLa (cervical cancer) cells in response to co-culturing with activated PBMCs, treatment with conditioned media collected from activated PBMCs, and direct treatment with recombinant IFN-y. As expected, IFN-y treatment induced dose-dependent upregulation of PD-L1 protein expression in both HCC38 and HeLa cell lines, whereas the effects of conditioned media and direct co-culturing with PBMCs resulted in more complex effects which are discussed. To determine if tumor cytoarchitecture influences responsiveness to the PBMCs, biomarker expression was measured from cultures grown in both traditional adherent monolayers and in 3D spheroid cultures using Ultra-Low Attachment (ULA) microplates. Cellular health and proliferation was assessed by measuring ATP concentration with ATPlite luminescence assays and cellular imaging with viability dyes. The upregulation of PD-L1 expression was observed to be largely independent of cellular proliferation and is further shown to be dependent on signaling through the JAK/STAT pathway by probing with AlphaLISA SureFire phosphorylation assays. These data illustrate and address some of the challenges in developing a biologically relevant culture system for examining the complex mechanisms involved in tumor evasion of the innate immune response. Citation Format: Jeanine M. Hinterneder, Jen Carlstrom, Adam Carlson, Dawn Nida. Rapid, no-wash measurement of immune checkpoint molecules expression induced by interaction with peripheral blood mononuclear cells in breast and cervical cancer cell models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4671.

FOXO Transcription Factors Rewire Metabolism in U87MG Glioblastoma Cells

Evolutionarily‐conserved FOXO transcription factors reside in the nucleus of glioblastoma and basal breast cancer cells that harbor constitutive PI3K Pathway output. Nuclear localization of FOXO factors in this setting challenges canonical PI3K models, as this pathway typically induces FOXO localization to the cytoplasm. Simultaneous PI3K activation and nuclear FOXO have also been observed in a set of diffuse large B cell lymphomas and embryonic stem cells. To interrogate the function of nuclear‐localized FOXO transcription factors in glioblastoma and basal breast cancer cell lines, we have constructed genetic models using CRISPR Cas9 genome editing technology. Our evidence indicates that FOXO factors drive metabolic changes in these cancers. FOXO disruption mutants showed increased mitochondrial membrane potential by TMRE staining and subsequent flow cytometric analyses. FOXO factors promote PEPCK gene expression to likely impact carbon utilization. These findings suggest that the nuclear localization of FOXO transcription factors causes metabolic rewiring in glioblastoma and basal breast cancer.Support or Funding InformationThis work was supported by HHMI 52007568 (N.V.), USDA Step 2 2015‐38422‐24061(A.L., R.G,), USDA H.S.I. 2016‐38422‐25760 (M.K. and E.M), NIH 1SC3GM11666901 (R.G.), UTRGV College of Sciences (COS) Seed Grant (M.K.), and NSF Advance 1209210 (M.K).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Breast Cancer Spheroids Reveal a Differential Cancer Stem Cell Response to Chemotherapeutic Treatment

An abnormal multicellular architecture is a defining characteristic of breast cancer and, yet, most in vitro tumor models fail to recapitulate this architecture or accurately predict in vivo cellular responses to therapeutics. The efficacy of two front-line chemotherapeutic agents (paclitaxel and cisplatin) are described within three distinct in vitro models employing the triple-negative basal breast cancer cell line MDA-MB-231 and the luminal breast cancer cell line MCF7: a) a 3D collagen embedded multicellular spheroid tumor model, which reflects the architecture and cellular heterogeneity of tumors in vivo; b) a 3D collagen model with a single cell-type diffusely embedded; and c) a 2D monolayer. The MDA-MB-231 embedded spheroid tumor model exhibited the most robust response to chemotherapeutic treatment, and possessed the greatest cancer stem cell (CSC) content. CSC-related genes are elevated across all MDA-MB-231 in vitro models following paclitaxel treatment, indicating that paclitaxel enrichment of chemoresistant CSCs is less dependent on microenvironmental tumor structure, while cisplatin showed a more context-dependent response. In the MCF7 cell models a context-dependent response is observed with paclitaxel treatment increasing the CSC related genes in the 2D monolayer and 3D diffuse models while cisplatin treatment afforded an increase in ALDH1A3 expression in all three models.

Prognostic and functional role of subtype‐specific tumor–stroma interaction in breast cancer

None of the clinically relevant gene expression signatures available for breast cancer were specifically developed to capture the influence of the microenvironment on tumor cells. Here, we attempted to build subtype‐specific signatures derived from an in vitro model reproducing tumor cell modifications after interaction with activated or normal stromal cells. Gene expression signatures derived from HER2+, luminal, and basal breast cancer cell lines (treated by normal fibroblasts or cancer‐associated fibroblasts conditioned media) were evaluated in clinical tumors by in silico analysis on published gene expression profiles (GEPs). Patients were classified as microenvironment‐positive (μENV+ve), that is, with tumors showing molecular profiles suggesting activation by the stroma, or microenvironment‐negative (μENV−ve) based on correlation of their tumors' GEP with the respective subtype‐specific signature. Patients with estrogen receptor alpha (ER)+/HER2−/μENV+ve tumors were characterized by 2.5‐fold higher risk of developing distant metastases (HR = 2.546; 95% CI: 1.751–3.701, P = 9.84E‐07), while μENV status did not affect, or only suggested the risk of distant metastases, in women with HER2+ (HR = 1.541; 95% CI: 0.788–3.012, P = 0.206) or ER‐/HER2− tumors (HR = 1.894; 95% CI: 0.938–3.824; P = 0.0747), respectively. In ER+/HER2− tumors, the μENV status remained significantly associated with metastatic progression (HR = 2.098; CI: 1.214–3.624; P = 0.00791) in multivariable analysis including size, age, and Genomic Grade Index. Validity of our in vitro model was also supported by in vitro biological endpoints such as cell growth (MTT assay) and migration/invasion (Transwell assay). In vitro‐derived gene signatures tracing the bidirectional interaction with cancer activated fibroblasts are subtype‐specific and add independent prognostic information to classical prognostic variables in women with ER+/HER2− tumors.

Oncosis and apoptosis induction by activation of an overexpressed ion channel in breast cancer cells

The critical role of calcium signalling in processes related to cancer cell proliferation and invasion has seen a focus on pharmacological inhibition of overexpressed ion channels in specific cancer subtypes as a potential therapeutic approach. However, despite the critical role of calcium in cell death pathways, pharmacological activation of overexpressed ion channels has not been extensively evaluated in breast cancer. Here we define the overexpression of transient receptor potential vanilloid 4 (TRPV4) in a subgroup of breast cancers of the basal molecular subtype. We also report that pharmacological activation of TRPV4 with GSK1016790A reduced viability of two basal breast cancer cell lines with pronounced endogenous overexpression of TRPV4, MDA-MB-468 and HCC1569. Pharmacological activation of TRPV4 produced pronounced cell death through two mechanisms: apoptosis and oncosis in MDA-MB-468 cells. Apoptosis was associated with PARP-1 cleavage and oncosis was associated with a rapid decline in intracellular ATP levels, which was a consequence of, rather than the cause of, the intracellular ion increase. TRPV4 activation also resulted in reduced tumour growth in vivo. These studies define a novel therapeutic strategy for breast cancers that overexpress specific calcium permeable plasmalemmal ion channels with available selective pharmacological activators.