Basal-like Breast Cancer Cell Lines Research Articles

MiR-424-5p regulates cell proliferation, migration and invasion by targeting doublecortin-like kinase 1 in basal-like breast cancer

Our previous study has showed doublecortin like kinase 1 (DCLK1) serves as an oncogene to regulate basal-like breast cancer cell proliferation, migration and invasion, and is associated with malignant status and poor prognosis. The aim of this study is to identify microRNAs (miRNAs), which target DCLK1 to regulate basal-like breast cancer cell proliferation, migration and invasion. In our results, we observed that miR-424-5p expression was decreased in basal-like breast cancer tissues and cell lines. Furthermore, we found 3’-UTR of DCLK1 had binding site of miR-424-5p based on microRNA target databases, and there was an inverse correlation between miR-424-5p and DCLK1 in basal-like breast cancer tissues. Moreover, we confirmed miR-424-5p directly targeted to 3’-UTR of DCLK1 through luciferase reporter assay, and miR-424-5p negatively regulated DCLK1 mRNA and protein expressions through qRT-PCR and western blot. The gain-of-function studies showed that miR-424-5p suppressed basal-like breast cancer cell proliferation, migration and invasion. The rescued-function studies suggested up-regulation of DCLK1 could rescue inhibition of miR-424-5p mimics in the regulation of basal-like breast cancer cell proliferation, migration and invasion. Finally, low-expression of miR-424-5p was associated with advanced clinical stage, large tumor size, more metastatic lymph nodes, present distant metastasis and poor histological grade in basal-like breast cancer patients. In conclusion, miR-424-5p is a tumor suppressive microRNA to regulate tumor cell proliferation, migration and invasion via binding to the functional target DCLK1, and associated with malignant status in basal-like breast cancer.

Evaluation of FOXC1 as a therapeutic target for basal-like breast cancer.

Basal-like breast cancer (BLBC) is a malignant carcinoma with aggressive motility and rapid growth. Accounting for 15% of breast cancers, BLBC often exhibits a poor prognosis and tends to metastasize to the brain and lungs. Because most BLBC display a triple-negative phenotype (ER-, PR-, and HER2-), conventional cytotoxic chemotherapy remains the only treatment option despite poor success and high rate of relapse. The overexpression of the forkhead-box transcription factor C1 (FOXC1) was recently identified as a biomarker of BLBC. Increased expression of FOXC1 was linked to excessive mobility and growth of BLBC cell lines, suggesting FOXC1 as a therapeutic target. In this study, siRNA-mediated knockdown of FOXC1 was confirmed to decrease the proliferation rate, migration, and invasion in a model BLBC-like cell line (4T1). 4T1 and 4T1-∆FOXC1 cells lacking FOXC1 expression (generated by CRISPR/Cas9) were used to evaluate the effects of FOXC1 expression in an orthotopic murine model of BLBC. No statistically significant difference in tumor volume was observed between 4T1 and 4T1-∆FOXC1 tumors. Furthermore, tumors metastasized to the liver and lungs to a similar degree regardless of FOXC1 expression. These data suggest that, despite positive results in vitro, FOXC1 may not be a promising therapeutic target for BLBC.

Assessment of cytosolic free calcium changes during ceramide-induced cell death in MDA-MB-231 breast cancer cells expressing the calcium sensor GCaMP6m

Alterations in Ca2+ signaling can regulate key cancer hallmarks such as proliferation, invasiveness and resistance to cell death. Changes in the regulation of intracellular Ca2+ and specific components of Ca2+ influx are a feature of several cancers and/or cancer subtypes, including the basal-like breast cancer subtype, which has a poor prognosis. The development of genetically encoded calcium indicators, such as GCaMP6, represents an opportunity to measure changes in intracellular free Ca2+ during processes relevant to breast cancer progression that occur over long periods (e.g. hours), such as cell death. This study describes the development of a MDA-MB-231 breast cancer cell line stably expressing GCaMP6m. The cell line retained the key features of this aggressive basal-like breast cancer cell line. Using this model, we defined alterations in relative cytosolic free Ca2+ ([Ca2+]CYT) when the cells were treated with C2-ceramide. Cell death was measured simultaneously via assessment of propidium iodide permeability. Treatment with ceramide produced delayed and heterogeneous sustained increases in [Ca2+]CYT. Where cell death occurred, [Ca2+]CYT increases preceded cell death. The sustained increases in [Ca2+]CYT were not related to the rapid morphological changes induced by ceramide. Silencing of the plasma membrane Ca2+ ATPase isoform 1 (PMCA1) was associated with an augmentation in ceramide-induced increases in [Ca2+]CYT and also cell death. This work demonstrates the utility of GCaMP6 Ca2+ indicators for investigating [Ca2+]CYT changes in breast cancer cells during events relevant to tumor progression, which occur over hours rather than minutes.

Establishment of two basal-like breast cancer cell lines with extremely low tumorigenicity from Taiwanese premenopausal women

The research of carcinogenetic mechanisms of breast cancer in different ethnic backgrounds is an interesting field, as clinical features of breast cancers vary among races. High premenopausal incidence is distinctive in East-Asian breast cancer. However, human cell lines derived from Asian primary breast tumor are rare. To provide alternative cell line models with a relevant genetic background, we aimed to establish breast cancer cell lines from Taiwanese patients of Han-Chinese ethnicity. Fresh tissue from mammary tumors were digested into organoids, plated and grown in basal serum-free medium of human mammary epithelial cells (HuMEC) with supplements. Cells were further enriched by positive selection with CD326 (epithelial cell adhesion molecule; EpCAM)-coated micro-magnetic beads. Two breast cancer cell lines derived from premenopausal women were successfully established by this method, and named Chang-Gung Breast Cancer 01 (CGBC 01) and 02 (CGBC 02). These two cell lines had a similar phenotype with weak expression of estrogen receptor (ER), progesterone receptor (PR), and without amplification of receptor tyrosine protein kinase erbB-2 (HER2/neu). Genome-wide Single Nucleotide Polymorphism (SNP) array showed multiple copy number alterations in both cell lines. Based on gene expression profiles, CGBC 01 and 02 were clustered into basal-like subtype with reference to the breast cancer cell line gene expression database. The tumorigenicity of both cell lines was extremely low in both anchorage-independence assay and transplantation into the mammary fat pads of nude mice. CGBC 01 and CGBC 02 are low tumorigenic breast cancer cell lines, established from Han-Chinese premenopausal breast cancer patients, which serve as in vitro models in studying the biological features of Asian breast cancer.

Proteomic analysis defines kinase taxonomies specific for subtypes of breast cancer.

Multiplexed small molecule inhibitors covalently bound to Sepharose beads (MIBs) were used to capture functional kinases in luminal, HER2-enriched and triple negative (basal-like and claudin-low) breast cancer cell lines and tumors. Kinase MIB-binding profiles at baseline without perturbation proteomically distinguished the four breast cancer subtypes. Understudied kinases, whose disease associations and pharmacology are generally unexplored, were highly represented in MIB-binding taxonomies and are integrated into signaling subnetworks with kinases that have been previously well characterized in breast cancer. Computationally it was possible to define subtypes using profiles of less than 50 of the more than 300 kinases bound to MIBs that included understudied as well as metabolic and lipid kinases. Furthermore, analysis of MIB-binding profiles established potential functional annotations for these understudied kinases. Thus, comprehensive MIBs-based capture of kinases provides a unique proteomics-based method for integration of poorly characterized kinases of the understudied kinome into functional subnetworks in breast cancer cells and tumors that is not possible using genomic strategies. The MIB-binding profiles readily defined subtype-selective differential adaptive kinome reprogramming in response to targeted kinase inhibition, demonstrating how MIB profiles can be used in determining dynamic kinome changes that result in subtype selective phenotypic state changes.

Assessment of CXC ligand 12-mediated calcium signalling and its regulators in basal-like breast cancer cells.

CXC ligand (L)12 is a chemokine implicated in the migration, invasion and metastasis of cancer cells via interaction with its receptors CXC chemokine receptor (CXCR)4 and CXCR7. In the present study, CXCL12-mediated Ca2+ signalling was compared with two basal-like breast cancer cell lines, MDA-MB-231 and MDA-MB-468, which demonstrate distinct metastatic potential. CXCL12 treatment induced Ca2+ responses in the more metastatic MDA-MB-231 cells but not in the less metastatic MDA-MB-468 cells. Assessment of mRNA levels of CXCL12 receptors and their potential modulators in both cell lines revealed that CXCR4 and CXCR7 levels were increased in MDA-MB-231 cells compared with MDA-MB-468 cells. Cluster of differentiation (CD)24, the negative regulator of CXCL12 responses, demonstrated increased expression in MDA-MB-468 cells compared with MDA-MB-231 cells, and the two cell lines expressed comparable levels of hypoxia-inducible factor (HIF)2α, a CXCR4 regulator. Induction of epithelial-mesenchymal transition (EMT) by epidermal growth factor exhibited opposite effects on CXCR4 mRNA levels compared with hypoxia-induced EMT. Neither EMT inducer exhibited an effect on CXCR7 expression, however hypoxia increased HIF2α expression levels in MDA-MB-468 cells. Analysis of the gene expression profiles of breast tumours revealed that the highest expression levels of CXCR4 and CXCR7 were in the Claudin-Low molecular subtype, which is markedly associated with EMT features.

Antioxydation And Cell Migration Genes Are Identified as Potential Therapeutic Targets in Basal-Like and BRCA1 Mutated Breast Cancer Cell Lines.

Basal-like breast cancers are among the most aggressive cancers and effective targeted therapies are still missing. In order to identify new therapeutic targets, we performed Methyl-Seq and RNA-Seq of 10 breast cancer cell lines with different phenotypes. We confirmed that breast cancer subtypes cluster the RNA-Seq data but not the Methyl-Seq data. Basal-like tumor hypermethylated phenotype was not confirmed in our study but RNA-Seq analysis allowed to identify 77 genes significantly overexpressed in basal-like breast cancer cell lines. Among them, 48 were overexpressed in triple negative breast cancers of TCGA data. Some molecular functions were overrepresented in this candidate gene list. Genes involved in antioxydation, such as SOD1, MGST3 and PRDX or cadherin-binding genes, such as PFN1, ITGB1 and ANXA1, could thus be considered as basal like breast cancer biomarkers. We then sought if these genes were linked to BRCA1, since this gene is often inactivated in basal-like breast cancers. Nine genes were identified overexpressed in both basal-like breast cancer cells and BRCA1 mutated cells. Amongst them, at least 3 genes code for proteins implicated in epithelial cell migration and epithelial to mesenchymal transition (VIM, ITGB1 and RhoA).Our study provided several potential therapeutic targets for triple negative and BRCA1 mutated breast cancers. It seems that migration and mesenchymal properties acquisition of basal-like breast cancer cells is a key functional pathway in these tumors with a high metastatic potential.

Formin Proteins FHOD1 and INF2 in Triple-Negative Breast Cancer: Association With Basal Markers and Functional Activities

Basal-like breast cancer is an aggressive form of breast cancer with limited treatment options. The subgroup can be identified immunohistochemically, by lack of hormone receptor expression combined with expression of basal markers such as CK5/6 and/or epidermal growth factor receptor (EGFR). In vitro, several regulators of the actin cytoskeleton are essential for efficient invasion of basal-like breast cancer cell lines. Whether these proteins are expressed in vivo determines the applicability of these findings in clinical settings. The actin-regulating formin protein FHOD1 participates in invasion of the triple-negative breast cancer cell line MDA-MB-231. Here, we measure the expression of FHOD1 protein in clinical triple-negative breast cancers by using immunohistochemistry and further characterize the expression of another formin protein, INF2. We report that basal-like breast cancers frequently overexpress formin proteins FHOD1 and INF2. In cell studies using basal-like breast cancer cell lines, we show that knockdown of FHOD1 or INF2 interferes with very similar processes: maintenance of cell shape, migration, invasion, and proliferation. Inhibition of EGFR, PI3K, or mitogen-activated protein kinase activity does not alter the expression of FHOD1 and INF2 in these cell lines. We conclude that the experimental studies on these formins have implications in the clinical behavior of basal-like breast cancer.

Chondroitin sulfate–mediated N-cadherin/β-catenin signaling is associated with basal-like breast cancer cell invasion

Tumor metastasis involves cancer cell invasion across basement membranes and interstitial tissues. The initial invasion step consists of adherence of the tumor cell to the extracellular matrix (ECM), and this binding transduces a variety of signals from the ECM to the tumor cell. Accordingly, it is critical to establish the mechanisms by which extracellular cues influence the intracellular activities that regulate tumor cell invasion. Here, we found that invasion of the basal-like breast cancer cell line BT-549 is enhanced by the ECM component chondroitin sulfates (CSs). CSs interacted with and induced proteolytic cleavage of N-cadherin in the BT-549 cells, yielding a C-terminal intracellular N-cadherin fragment that formed a complex with β-catenin. Of note, the cleavage of N-cadherin increased cytoplasmic and nuclear β-catenin levels; induced the matrix metalloproteinase 9 (MMP9) gene, a target of β-catenin nuclear signaling; and augmented the invasion potential of the cells. We also found that CS-induced N-cadherin proteolysis requires caveolae-mediated endocytosis. An inhibitor of that process, nystatin, blocked both the endocytosis and proteolytic cleavage of N-cadherin induced by CS and also suppressed BT-549 cell invasion. Knock-out of chondroitin 4-O-sulfotransferase-1 (C4ST-1), a key CS biosynthetic enzyme, suppressed activation of the N-cadherin/β-catenin pathway through N-cadherin endocytosis and significantly decreased BT-549 cell invasion. These results suggest that CSs produced by C4ST-1 might be useful therapeutic targets in the management of basal-like breast cancers.

Discovery of internalizing antibodies to basal breast cancer cells.

We present a strategy to discover recombinant monoclonal antibodies (mAbs) to specific cancers and demonstrate this approach using basal subtype breast cancers. A phage antibody library was depleted of antibodies to common cell surface molecules by incubation with luminal breast cancer cell lines, and then selected on a single basal-like breast cancer cell line (MDA-MB-231) for binding associated receptor-mediated endocytosis. Additional profiling against two luminal and four basal-like cell lines revealed 61 unique basal-specific mAbs from a pool of 1440 phage antibodies. The unique mAbs were further screened on nine basal and seven luminal cell lines to identify those with the greatest affinity, specificity, and internalizing capability for basal-like breast cancer cells. Among the internalizing basal-specific mAbs were those recognizing four transmembrane receptors (EphA2, CD44, CD73 and EGFR), identified by immunoprecipitation-mass spectrometry and yeast-displayed antigen screening. Basal-like breast cancer expression of these four receptors was confirmed using a bioinformatic approach, and expression microarray data on 683 intrinsically subtyped primary breast tumors. This overall approach, which sequentially employs phage display antibody library selection, antigen identification and bioinformatic confirmation of antigen expression by cancer subtypes, offers efficient production of high-affinity mAbs with diagnostic and therapeutic utility against specific cancer subtypes.

Human antigen R-regulated CCL20 contributes to osteolytic breast cancer bone metastasis

Breast cancer mainly spreads to bone, causing decreased survival of patient. Human antigen R (HuR) and chemokines are important molecules associated with mRNA stability and cell-cell interaction in cancer biology. Here, HuR knockdown inhibited bone metastasis and osteolysis of metastatic breast cancer cells in mice and HuR expression promoted the metastatic ability of cancer cells via CCL20 and GM-CSF. In contrast with the findings for GM-CSF, ELAVL1 and CCL20 expressions were markedly increased in breast tumor tissues and ELAVL1 expression showed a strong positive correlation with CCL20 expression in breast cancer subtypes, particularly the basal-like subtype. Metastasis-free survival and overall survival were decreased in the breast cancer patients with high CCL20 expression. We further confirmed the role of CCL20 in breast cancer bone metastasis. Intraperitoneal administration of anti-CCL20 antibodies inhibited osteolytic breast cancer bone metastasis in mice. Treatment with CCL20 noticeably promoted cell invasion and the secretion of MMP-2/9 in the basal-like triple-negative breast cancer cell lines, not the luminal. Moreover, CCL20 elevated the receptor activator of nuclear factors kappa-B ligand/osteoprotegerin ratio in breast cancer and osteoblastic cells and mediated the crosstalk between these cells. Collectively, HuR-regulated CCL20 may be an attractive therapeutic target for breast cancer bone metastasis.

Development and cytotoxic response of two proliferative MDA-MB-231 and non-proliferative SUM1315 three-dimensional cell culture models of triple-negative basal-like breast cancer cell lines.

Triple-Negative Basal-Like tumors, representing 15 to 20% of breast cancers, are very aggressive and with poor prognosis. Targeted therapies have been developed extensively in preclinical and clinical studies to open the way for new treatment strategies. The present study has focused on developing 3D cell cultures from SUM1315 and MDA-MB-231, two triple-negative basal-like (TNBL) breast cancer cell lines, using the liquid overlay technique. Extracellular matrix concentration, cell density, proliferation, cell viability, topology and ultrastructure parameters were determined. The results showed that for both cell lines, the best conditioning regimen for compact and homogeneous spheroid formation was to use 1000 cells per well and 2% Geltrex®. This conditioning regimen highlighted two 3D cell models: non-proliferative SUM1315 spheroids and proliferative MDA-MB-231 spheroids. In both cell lines, the comparison of 2D vs 3D cell culture viability in the presence of increasing concentrations of chemotherapeutic agents i.e. cisplatin, docetaxel and epirubicin, showed that spheroids were clearly less sensitive than monolayer cell cultures. Moreover, a proliferative or non-proliferative 3D cell line property would enable determination of cytotoxic and/or cytostatic drug activity. 3D cell culture could be an excellent tool in addition to the arsenal of techniques currently used in preclinical studies.

Establishment and characterization of a triple negative basal-like breast cancer cell line with multi-drug resistance.

Triple-negative breast carcinoma (TNBC) is one of the most aggressive subtypes of breast cancer and is associated with an unfavorable prognosis. The management of TNBC is currently based on the use of classical cytotoxic drugs, i.e., anthracyclines and/or microtubule-binding agents (TBAs). However, conventional chemotherapy is not always effective in these tumors and a systemic relapse is often observed, potentially due to the development of multi-drug resistance (MDR). Therefore, an improved understanding of MDR mechanisms may improve the therapeutic strategies for TNBC. In the present study, a paclitaxel-resistant (TxR) breast cancer cell subline of HCC1806 TNBC cells was established and characterized. The resistance index of this subline was calculated according to the IC50 of HCC1806-TxR relative to the parental HCC1806 cells (16.86-fold). TxR-cells also exhibited cross-resistance to vinblastin, doxorubicin and etoposide (~14-, ~4- and ~3-fold, respectively). As assessed with reverse transcription-quantitative polymerase chain reaction, TxR-resistant cells exhibited the upregulated expression of a number of multidrug resistance-associated genes, including MDR-1, MRP-1, -5, -6 and YB-1. The TxR cells also exhibited an increased expression of MDR-related proteins including MDR1 and MRP-1, which led to a substantial increase (5.4-fold) of the paclitaxel efflux from TxR-cells. In addition, the pro-apoptotic protein Fas was downregulated, whereas the anti-apoptotic Bcl-2 was upregulated, in TxR-cells. This may explain why a reduced extent of apoptosis was observed when TxR cells were exposed to TBAs and topoisomerase type II inhibitors, relative to the parental HCC1806 cells. Thus, the HCC1806-TxR cell line may serve as an appropriate model for the analysis of chemoresistance mechanisms in TNBCs, and for the investigation of novel anticancer agents for overcoming MDR-mediated mechanisms in TNBC.

Abstract 5552: Differential proteomic responses of luminal-A and basal-like breast cancer cell lines during growth inhibition induced by co-culture with agarose encapsulated murine renal adenocarcinoma (RENCA) cells

Abstract The existence of a tumor growth regulatory network that is conserved across tumor types and species has been hypothesized. Our evidence for this regulatory network is derived from studies demonstrating tumor growth inhibition by agarose encapsulated cancer cells (cancer macrobeads). The ability of encapsulated, thus growth restricted cells, to inhibit freely growing cancer cells has been shown when the encapsulated cells are murine (RENCA) or human (e.g., J82). Clinical trials are underway (NCT01053013, NCT02046174). We have shown that RENCA macrobeads release >10 known tumor inhibitory proteins targeting several signaling pathways including Akt/PI3. To better understand the mechanism(s) of growth inhibition, we used a systems biology approach to identify protein profiles and interactions from macrobead-treated human breast carcinoma cell lines that are either mildly aggressive (MCF7) or highly aggressive (MDA-MB231 [MDA]). Target cells were co-cultured with RENCA macrobeads or left untreated for 5 days. Lysates of target cells were prepared in Laemmli buffer, frozen, and sent for MS/MS. Samples were run in duplicate, protein intensities normalized, and the treated/untreated ratio log10 transformed to get a normal distribution. Protein profiles were analyzed using Key Pathway Analysis (KPA) and Metacore software. Growth inhibition by RENCA macrobeads was confirmed (MCF7 25%; MDA 57%). KPA of MCF7 proteins showed upregulation of ubiquitin pathways involved in degradation of misfolded proteins. Stress induced apoptosis and DNA damage (which correlates with the growth inhibition) were the top 2 pathways upregulated using Pathway and Process Enrichment analysis. This is in line with a strong epigenetic gene deregulation (Metacore). Transcription Factor and Network analyses show upregulation of CREB, A2MR and ATF3 networks. Also, Regulated Network analysis suggests a role for A2MR or RAGE in the inhibition of proliferation and increased apoptosis. Macrobead-treated MDA cells showed no significant changes using KPA. Pathway, Network and Process enrichment (Metacore) showed 5 of the top 10 upregulated processes were associated with cytoskeleton remodeling. Cell cycle and protein folding processes were also upregulated. Transcription factors associated with this response were similar to that of MCF7. RAGE again appears in many of the pathways as indicated by Network and Regulated Network analysis. The protein response to RENCA macrobeads differs for these 2 cell lines. Proteins related to apoptosis are upregulated in the MCF7 cells whereas MDA have a preference for cytoskeleton remodeling. These data support the hypothesis that distinctive tumors may respond differently to RENCA macrobead exposure at both an epigenetic and protein level, nonetheless resulting in growth inhibition. Citation Format: Melissa A. Laramore, Peter James, Prithy C. Martis, Atira Dudley, Lawrence S. Gazda, Carl A. Borrebaeck, Barry H. Smith. Differential proteomic responses of luminal-A and basal-like breast cancer cell lines during growth inhibition induced by co-culture with agarose encapsulated murine renal adenocarcinoma (RENCA) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5552. doi:10.1158/1538-7445.AM2017-5552

Abstract 3697: The impact of HER3 signaling mediated PD-L1 regulation in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is still difficult to treat partly because of lacking specific target. Although 50-70% of TNBC expresses EGFR, it is less sensitive to the treatment of EGFR inhibition for TNBC as compared to the efficacy of HER2 inhibition for HER2-positive breast cancer. Several phase II study on EGFR blockade treatment has been reported, however it has not applied in a clinical setting yet. It was reported that residual tumors after treatment with EGFR-targeted antibodies showed increased HER3 abundance leading to EGFR/HER3 receptor dimerization. The signals of HER3/EGFR dimerization to PI3K/AKT/mTOR pathways are thought to be involved in cancer survival, proliferation and also up-regulation of PD-L1 expression. Thus, we hypothesized that up-regulation of HER3 signal caused by anti-cancer treatment might induce PD-L1 expression and inhibit host anti-tumor immunity. In this study, we tested the relationships between HER3 signal and PD-L1 expression by using three basal-like breast cancer cell line; MDA-MB-231, HCC70, and MDA-MB-468. MDA-MB-231 is HER3-negative, and HCC70 and MDA-MB-468 are HER3-positive cell lines. We added neureglin 1 (NRG1: HER3 ligand) to those three cell lines and analyzed PD-L1 expression of protein by flowcytometry and mRNA by qRT-PCR. Both protein and mRNA level of PD-L1 on HCC70 and MDA-MB-468 treated with NRG1 are increased as compared with those without NRG1 while there was no change of PD-L1 expression of MDA-MB-231 either with or without NRG1. In order to confirm the significance of potential treatment target of HER3, we evaluated HER3 expression in biopsy samples by immunohistochemistry before neoadjuvant chemotherapy (NAC) including all phenotypes. Thirteen pathological complete response (pCR) cases after NAC and 6 non pCR cases were included. We scored the HER3 stainability from 0 to 3 and found that non pCR cases showed significantly higher HER3 score than pCR cases (84.6% and 33% respectively, p=0.0149). Although further study is needed, these results suggest that HER3 signal possibly regulates PD-L1 expression in HER3-positive basal-like breast cancer and treatment with anti-HER3 targeting therapy combination with an immune checkpoint inhibition therapy for HER3 positive NAC resistant patients might be warranted. Note: This abstract was not presented at the meeting. Citation Format: Ayane Yamaguchi, Eiji Suzuki, Kosuke Kawaguchi, Mariko Nishie, Moe Tsuda, Takeshi Kotake, Masakazu Toi. The impact of HER3 signaling mediated PD-L1 regulation in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3697. doi:10.1158/1538-7445.AM2017-3697

Abstract 1265: Mechanisms underlying genomic integrity in breast cancer-derived dormant circulating tumor cells

Abstract Patients with metastatic breast cancer have a five-year survival rate of 22% compared to 99% in patients with cancer localized to the breast. This disparity in clinical outcome is at least in part caused by the lack of diagnostic tools for early detection of metastatic recurrence. This can be especially valid for breast cancer patients having brain metastases (BCBM), 10-20% of whom are diagnosed only at autopsy. The early detection of circulating tumor cells (CTCs) in peripheral blood during the metastatic cascade provides a window of opportunity to prevent BCBM onset. However, to target CTCs causing BCBM, one must dissect mechanisms that allow CTCs to retain their brain metastatic potency while remaining dormant in circulation. We hypothesized that hyperactive mechanisms of DNA repair preserve the genomic make-up of dormant CTCs allowing them to maintain over time the potential for BCBM competence. Comprehensive analyses of breast cancer patient-derived CTC transcriptomes showed that CTC gene signatures closely relate to basal-like breast cancer cell lines rather than the molecular subtype of the primary tumor. However, unlike actively cycling cell lines, a large proportion of CTCs were mitotically dormant (Ki67-/PCNA-) with low overall mRNA expression and transcriptional activity. Upon systemic injection of patient-derived common lineage-depleted cells into immuno-compromised mice, the majority of CTCs were found at metastatic sites in a state of mitotic/metabolic dormancy. Second, dormant CTCs had a lower incidence of double-strand DNA breaks (DSB) than proliferating cells as assessed by Serine139 phosphorylation status of gamma H2AX. Third, we discovered that the human telomere-associated protein RIF1, a mediator of alternative non-homologous end joining repair of DNA DSB interplaying with BRCA1/2, was significantly up-regulated in the Ki67-/PCNA- CTC population. RIF1/BRCA1 foci formation was confirmed in areas of DNA damage in these cells. Lastly, shRNA-mediated RIF1 knockdown promoted dormant cell populations to become more susceptible to UV and bleomycin-induced DNA damage while activating p38 and ERK stress-response pathways as well as their phosphorylation ratio. Moreover, RIF1 knockdown in MDA-MB231BR clone attenuated its metastatic competence to the brain. Collectively, these findings suggest that RIF1 regulates the genomic integrity of dormant CTCs allowing them to survive over time while in circulation. Further elucidation of RIF1-mediated CTC pathways and its dynamics with BRCA1/2 may provide novel strategies for therapeutic intervention. This can be clinically useful in breast cancer patients that are yet to develop overt BCBM. Citation Format: Debasish Boral, Haowen N. Liu, Wei Yin, Monika Vishnoi, Marc L. Sprouse, Jenny C. Chang, Jean Paul Thiery, Scamardo Antonio, David S. Hong, Dario Marchetti. Mechanisms underlying genomic integrity in breast cancer-derived dormant circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1265. doi:10.1158/1538-7445.AM2017-1265

Abstract 4036: Induction of detrimental aneuploidy in basal breast cancer cells treated by MPS1 inhibitors in combination with paclitaxel

Abstract Basal-like breast cancer has received considerable attention in recent years, but despite all efforts, conventional chemotherapy remains the main therapeutic option in the clinic with taxane therapy, the most frequently prescribed treatment. A significant reason for the lack of effective therapeutics may be related to the high degree of heterogeneity and aneuploidy which is characteristic for basal-like breast cancer. A new approach in this field is the concept that cancer cells may be targeted by the introduction of a detrimental level of aneuploidy. Inhibition of one of the main spindle assembly checkpoint kinases, MPS1 is a rational choice to achieve this goal. We report here the identification of a synergistic effect between the novel, oral, highly potent and selective MPS1 inhibitor CCT289346 and paclitaxel. We demonstrate robust synergism in multiple basal-like breast cancer cell lines and provide mechanistic insights into how both compounds’ individual properties work together to maximise mitotic defects thereby increasing aneuploidy and cell death in a single round of mitosis. Additionally, in vivo studies in basal-like breast cancer xenograft models, including patient derived xenografts and systemic metastatic models, using clinically relevant doses of paclitaxel with well-tolerated doses of CCT289346, demonstrate significant benefit of combination of the two agents in comparison to paclitaxel alone. Citation Format: Simon J. Anderhub, Grace WY Mak, Amir Faisal, Katie Walsh, Mark Gurden, Liz Day, Paolo Innocenti, Hannah L. Woodward, Isaac M. Westwood, Kwai-Ming J. Cheung, Sebastien Naud, Angela Hayes, Gary Box, Alexis de Haven Brandon, Melanie R. Valenti, Lisa O’Fee, Harry Saville, Rosemary Burke, Rob LM van Montfort, Florence I. Raynaud, Suzanne A. Eccles, Julian Blagg, Swen Hoelder, Spiros Linardopoulos. Induction of detrimental aneuploidy in basal breast cancer cells treated by MPS1 inhibitors in combination with paclitaxel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4036. doi:10.1158/1538-7445.AM2017-4036

The RB-IL-6 axis controls self-renewal and endocrine therapy resistance by fine-tuning mitochondrial activity.

Retinoblastoma (RB) protein inactivation during tumor progression is often associated with acquisition of immature phenotypes and resistance to therapy. Determination of an RB inactivation signature in a context of gaining undifferentiated phenotype in a p53-null sarcoma system revealed a critical role for interleukin (IL)-6. Using a Gene Set Enrichment Analysis (GSEA), we discovered that poorly differentiated breast cancers are enriched for this RB inactivation signature. Accelerated IL-6 secretion following RB inactivation in an RB-intact luminal-type breast cancer cell line MCF-7 promoted a positive feed forward loop between IL-6 and STAT3 driving tumor growth and endocrine therapy resistance. In addition, some of RB-intact basal-like type breast cancer cell lines exhibited a similar phenotype following RB depletion. The mechanism whereby RB inactivation increases IL-6 production in MCF-7 cells appeared to involve fatty acid oxidation (FAO)-dependent mitochondrial metabolism and c-Jun NH(2)-terminal kinase (JNK). In addition, IL-6, via STAT3-mediated feedback to mitochondria, autonomously adjusts mitochondrial superoxide to levels suitable to maintain stem cell-like activity. The gene expression profile of luminal-type breast cancer patients with low RB expression revealed high enrichment of genes involved in mitochondrial respiration and downstream targets of IL-6. These findings unveiled an unexpected strategy whereby RB suppresses malignant features of cancer cells through metabolic reprogramming and cell-autonomous inflammation.

Acetylated a-tubulin sense the change of extracellular matrix to regulate the invasion and metastasis of breast cancer

Objective To reveal acetylated a-tubulin acts as pressure sensors, sensing the changes in extracellular matrix to impact on the invasion and metastasis of breast cancer. Methods The acetylated alpha microtubule protein expressions were detected, and its relationship with invasion and metastasis in breast cancer clinical specimens and different molecular subtypes of breast cancer cell lines were analyzed. The expression of acetylated a-tubulin was interfered through chemical methods, and cell morphology and the change of the invasion and metastasis ability were detected under the condition of different matrix hardness. Results The expression of acetylated a-tubulin was highest in basal-like breast cancer tissue and cell lines, and was lowest expressed in the luminal B breast cancer tissue and the breast cancer cell lines. The expression of acetylated a-microtubule was positively correlated with the occurrence of breast cancer. Under the condition of soft substrate cultivation, cell polarization was declined, becoming the circular or oval shape. The acetylated a-tubulin caused reduction in cell polarity, accompanied with less invasion and metastasis ability. Conclusions The acetylated a-tubulin acts as pressure sensors, sensing the sclerosis of extracellular matrix in the process of tumorigenesis, promoting invasion and metastasis of breast cancer. Key words: Tubulin/ME; Extracellular matrix/ME; Breast neoplasms; Neoplasm invasiveness; Neoplasm metastasis

Abstract P3-07-08: Molecular subtype-specific expression of long noncoding RNA regulates proliferation of basal-like breast cancer cells

Abstract Background: Basal-like breast cancer (BLBC) disproportionally affects younger women and African American women, displaying aggressive clinical behavior with poor outcomes. BLBC is difficult to treat due to its lack of defined molecular targets, and with current treatment, patients often develop metastatic disease. A growing body of evidence points to long noncoding (lnc) RNAs as mediators of tumor progression, suggesting a new class of targets in cancer therapy. While dynamic changes in lncRNA expression are observed across many types of cancer, our understanding of their role in cancer biology is limited. The aim of this study is to identify lncRNAs differentially expressed in BLBC and to characterize the functional features of candidate lncRNA. Methods: Breast tumors and normal breast tissues were recruited from the U of Chicago Breast Cancer Tissue Bank under IRB approved protocols. Microarray profiling was done using Human LncRNA Array v3 (Arraystar) containing 30,586 lncRNAs and 26,109 mRNAs. Non-poly(A) RNAs were included with ribo-zero RNA-seq (Illumina HiSeq 4000). Molecular subtype of breast tumors was determined by PAM50 intrinsic classifier. In addition, CpG methylation of lncRNA promoter regions was analyzed with TCGA HumanMethylation450 Array data from 588 breast tissues. We performed in vitro studies using antisense oligonucleotide (ASO) knockdown and CRISPR-on overexpression in BLBC cell lines. Phenotypic consequences of perturbations of specific lncRNA expression were assessed using proliferation, apoptosis, cell cycle and homologous recombination assays. Results: Microarray profiling of breast tissues from 30 African American women revealed a unique lncRNA signature in basal-like tumors compared to non-basal like tumors. Hundreds of lncRNAs were specifically expressed in BLBC, with >100-fold differences in some lncRNAs compared to tumors from other subtypes or normal breast tissues. Through statistical and in silico analyses we selected lncRNAs displaying increased expression in basal-like tumors (vs non-basal tumors) and BLBC cell lines (vs non-basal cell lines) with a trend of higher expression in cells isolated from women of African ancestry for functional studies. RNA-seq of 50 additional breast tissues recapitulated the distinct lncRNA clustering within the basal-like subtype. TCGA analysis of CpG islands in several lncRNA promoter regions revealed hypo-methylation when compared to non-BLBC, signifying subtype specific epigenetic regulation. ASO knockdown of candidate lncRNAs significantly increased apoptosis and decreased proliferation while CRISPR-on overexpression decreased cell sensitivity to doxorubicin treatment. Depletion of specific lncRNAs caused an increased proportion of cells in G2 phase of the cell cycle, indicating that overexpression of particular lncRNAs may contribute to aberrant cell cycle progression. Cis and trans-regulation of gene expression by lncRNAs is currently under investigation through RNA immunoprecipitation and genome-wide gene expression profiling. Conclusion: We propose that subtype specific lncRNAs function as onco-RNAs specific to BLBC, driving cellular proliferation and increasing resistance to DNA damaging chemotherapies through regulation of the cell cycle. Citation Format: Boatman SM, Han Y-J, Zhang J, Zheng Y, Yeh AC, Olopade OI. Molecular subtype-specific expression of long noncoding RNA regulates proliferation of basal-like breast cancer cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-08.