Low Metastatic Breast Cancer Cell Research Articles

CCL18 promotes breast cancer progression by exosomal miR-760 activation of ARF6/Src/PI3K/Akt pathway

The small GTPase ADP-ribosylation factor 6 (ARF6) mediates chemokine (C-C motif) ligand 18 (CCL18)-induced activation of breast cancer (BC) metastasis through its downstream effector AMAP1. However, the molecular mechanisms underlying CCL18 up-regulating ARF6 remain largely unclear. Here, microRNAs (miRNAs) that target ARF6 were predicted and selected in high metastatic BC cells treated with CCL18. Next, we assessed the role of exosomal miR-760 in vitro and in vivo. We further analyzed the expression of ARF6, AMAP1, and phosphorylated (p)-AMAP1 in tumor and adjacent normal tissues. We first observed that CCL18 increased the expression of ARF6 and p-AMAP1 and activated the Src/phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. ARF6 knockdown significantly impaired CCL18-induced malignant cellular behaviors and the Src/PI3K/Akt signaling pathway. Next, ARF6 was confirmed as a target gene of miR-760 in exosomes derived from CCL18-stimulated high metastatic BC cells. Moreover, recipient MCF-7 cells could effectively uptake these miR-760-rich exosomes that significantly promoted proliferation, tumor growth in vivo, migration, invasion, and chemoresistance by activating ARF6-mediated Src/PI3K/Akt signaling and the epithelial-mesenchymal transition (EMT) pathway. Together, our results support that exosomal miR-760 secreted by CCL18-stimulated high metastatic BC cells promoted the malignant behaviors in low metastatic BC cells by up-regulating the ARF6-mediated Src/PI3K/Akt signaling pathway.

Utilizing a high-throughput microdevice to study breast tumor cells clustering and metastasis

Circulating tumor cell (CTC) clusters, which are multicellular groups of CTCs, were recently suggested to had the greater potential of forming distal metastasis than single CTCs. However, our understanding of the forming of CTC clusters is still limited since there are few existing methods to study cancer cells aggregation kinetics, especially for a small number of cells. Herein we report a high-throughput miniaturized microwell-based cell aggregation-chip (AG-chip) to enable better characterize of the tumor cells clustering process. We successfully demonstrated the capability of the AG-chip in determining cell aggregation, and found that: (1) high metastatic breast cancer cells (MDA-MB-231 & MDA-MB-436) have stronger aggregation capacities than those low metastatic breast cancer cells (MCF-7 & SK-BR-3); (2) cells with similar aggregation ability were distinguished through the analysis of aggregation kinetics; (3) the detected aggregation ability can be used to indicate the metastatic potential of the cells; (4) the inhibition of integrins could regulate the cell clustering via blockage of cell adhesion or/and cell migration. This newly developed microdevice may promote further study of CTC clusters and metastasis.

Abstract 2403: A novel mechanism for regulation of breast cancer metastasis

Abstract Our major goal is to determine the role of Chromodomain-helicase-DNA-binding protein 7 during breast cancer (BC) metastasis. This protein is an ATP-dependent nucleosome remodeling factor and is critical for embryonic development in both animal models and human patients. Our most recent studies provide direct evidence suggesting its novel role in repressing BC metastasis. We found that this protein, but not the mRNA, is highly expressed in a low-metastatic breast cancer cell line (Mcf-7), while its expression is much reduced in metastatic cell lines (MDA-MB-468 and MDA-MB-231). Furthermore, this protein is predominantly localized in the cytoplasm of 468 and 231 cells, in contrast to its nuclear localization in Mcf-7 cells. In the cytoplasm of 468 and 231 cells, this protein is colocalized with a lysosome marker, suggesting that in high metastatic BC cells, this protein is exported to the cytoplasm to be degraded in lysosomes. Blocking the activity of lysosomes increased expression of this protein in high metastatic BC cell lines. This result suggests a mechanism accounting for the reduced expression of this protein in high metastatic BC cell lines. To further support the potential clinic relevance of our results, this protein is localized in the nucleus of almost all duct epithelial cells in normal breast tissues; however, in a large portion of epithelial cells of breast tumors, this protein is localized in the cytoplasm. Through reporter and mutagenesis analysis, we identified the nuclear exporting signal (NES) sequence responsible for exporting this protein into cytoplasm. In the next set of experiments, we attempted to determine the function of this gene in BC metastasis. We found that knocking down expression of this gene in 468 cell significantly increased their migration and invasion. We next applied the CRISPR genome editing technique to mutate the critical amino acids within the NES region in the endogenous gene locus and found that the amount of protein in the nucleus is significantly increased compared to wild type cells. Migration/invasion of these cells was also dramatically decreased. At the molecular level, we found that forced expression of this protein in nucleus reduced expression of epithelial markers and increased expression of epithelial-mesenchymal-transition markers. We are now applying ChIP-Seq and RNA-Seq approaches to determine direct regulatory network of this protein in 231 cells. We will further test whether this protein acts through modulating the epigenetic status of the enhancers/promoters to regulate expression of its target genes to repress BC metastasis. In summary, our data collectively suggest that Chromodomain-helicase-DNA-binding protein 7 is a novel epigenetic regulator to repress BC metastasis. To the best of our knowledge, our study represents the first to address the activity of this protein in BC. Note: This abstract was not presented at the meeting. Citation Format: Kenneth Jiao, Yin Peng, Lizhong Wang, Runhua Liu. A novel mechanism for regulation of breast cancer metastasis [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 2403. doi:10.1158/1538-7445.AM2017-2403

Pro-invasive stimuli and the interacting protein Hsp70 favour the route of alpha-enolase to the cell surface

Cell surface expression of alpha-enolase, a glycolytic enzyme displaying moonlighting activities, has been shown to contribute to the motility and invasiveness of cancer cells through the protein non-enzymatic function of binding plasminogen and enhancing plasmin formation. Although a few recent records indicate the involvement of protein partners in the localization of alpha-enolase to the plasma membrane, the cellular mechanisms underlying surface exposure remain largely elusive. Searching for novel interactors and signalling pathways, we used low-metastatic breast cancer cells, a doxorubicin-resistant counterpart and a non-tumourigenic mammary epithelial cell line. Here, we demonstrate by a combination of experimental approaches that epidermal growth factor (EGF) exposure, like lipopolysaccharide (LPS) exposure, promotes the surface expression of alpha-enolase. We also establish Heat shock protein 70 (Hsp70), a multifunctional chaperone distributed in intracellular, plasma membrane and extracellular compartments, as a novel alpha-enolase interactor and demonstrate a functional involvement of Hsp70 in the surface localization of alpha-enolase. Our results contribute to shedding light on the control of surface expression of alpha-enolase in non-tumourigenic and cancer cells and suggest novel targets to counteract the metastatic potential of tumours.

Abstract P6-01-14: Dissection of the Abl interactor 1 signaling in metastatic breast cancer cells

Abstract Abl interactor 1 (Abi1) is a key component of WAVE regulatory complex (WRC) that regulates actin cytoskeleton reorganization, membrane receptor signaling, and intracellular trafficking. Recent in vitro and in vivo studies as well as the studies of human patient samples suggest that Abi1 may play an important role in breast cancer (BC) metastasis. To determine if Abi1 is deregulated in metastatic breast cancer cells, we compared the expression of Abi1 in a panel of human breast cancer cell lines exhibiting distinct metastatic properties. In comparison with low-metastatic ER+/PR+ BC lines MCF-7 and T47D and a non-transforming mammary epithelial cell line MCF-10A, two ER-BC lines CN34 and MDA 231 and their sublines selected by high metastatic potential to bone (231Bo), brain (231Br and CN34Br), and lung (231LM2) show significantly reduced Abi1 protein levels, with greater reduction found in highly metastatic sublines. It is unlikely that the down-regulation of Abi1 expression in these metastatic BC cells occurs at mRNA level, because RT-PCR analysis shows no significant difference in Abi1 mRNA levels among the cells analyzed. Interestingly, despite a reduction in protein level, the tyrosine phosphorylation of Abi1 was increased in CN34, MDA231, and their metastatic sublines compared to low-metastatic BC cells. The tyrosine phosphorylation of Abi1 is stimulated by epidermal growth factor (EGF) in these cells and Abi1 is recruited to EGF receptor (EGFR) upon EGF stimulation. The deregulation of Abi1 in metastatic BC cells is mediated by Abl tyrosine kinases, as the inhibition of Abl family kinases by imatinib not only attenuates EGF-stimulated Abi1 tyrosine phosphorylation but also rescue the down-regulation of Abi1 protein in metastatic BC cells. These findings are consistent with a role of the EGFR-Abl-Abi1 signaling in BC metastasis. The studies suggest that this pathway may serve as a biomarker for diagnosis and/or prognosis of metastatic breast cancer. We have identified the tyrosine 213 in Abi1 as a major phosphorylation site stimulated by Abl kinases and have generated a unique conditional Abi1 depletion/re-expression system to dissect how the deregulation of the Abi1 signaling contributes to BC metastasis. Citation Format: Jiang P, Tang H, Hogan H, Tate S, Ryan W, Paul L, Liu X, Zonghan D. Dissection of the Abl interactor 1 signaling in metastatic 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 P6-01-14.

Human NUMB6 Induces Epithelial-Mesenchymal Transition and Enhances Breast Cancer Cells Migration and Invasion.

Mammalian NUMB is alternatively spliced generating four isoforms NUMB1-NUMB4 that can function as tumor suppressors. NUMB1-NUMB4 proteins, which normally determine how different cell types develop, are reduced in 21% of primary breast tumors. Our previous work has, however, indicated that two novel NUMB isoforms, NUMB5 and NUMB6 have the pro-oncogenic functions. Herein, we address a novel function of human NUMB isoform 6 (NUMB6) in promoting cancer cell migration and invasion. We found that NUMB6 induced expression of embryonic transcription factor Slug, which in turn actively repressed E-cadherin, prompting cells to undergo epithelial-mesenchymal transition (EMT). Low-metastatic breast cancer cells DB-7 stably expressing NUMB6, lost their epithelial phenotype, exhibited migratory and pro-invasive behavior, and ultimately elevated expression of mesenchymal markers. Among these markers, increased vimentin, β-catenin, and fibronectin expression elicited metalloproteinase 9 (MMP9) production. Our results revealed that NUMB6-DB-7 cells have significantly increased level of Akt1 and Akt2 phosphorylation. Therefore, antagonizing Akt signaling using a chemical inhibitor LY294002, we found that NUMB6-induced Slug expression was reduced, and ultimately accompanied with decreased cell migration and invasion. In summary, this study identified a novel molecular determinant of breast cancer progression, uncovering a potential oncogenic role for the NUMB6 protein in cancer cell migration and invasion, coupled to the maintenance of mesenchymal-like cells. J. Cell. Biochem. 118: 237-251, 2017. © 2016 Wiley Periodicals, Inc.

P2Y2 nucleotide receptor-mediated extracellular signal-regulated kinases and protein kinase C activation induces the invasion of highly metastatic breast cancer cells.

Tumor metastasis is considered the main cause of mortality in cancer patients, thus it is important to investigate the differences between high- and low-metastatic cancer cells. Our previous study showed that the highly metastatic breast cancer cell line MDA-MB-231 released higher levels of ATP and exhibited higher P2Y2R activity compared with the low-metastatic breast cancer cell line MCF-7. In addition, P2Y2R activation by ATP released from MDA-MB-231 cells induced hypoxia-inducible factor-1α expression, lysyl oxidase secretion and collagen crosslinking, generating a receptive microenvironment for pre-metastatic niche formation. Thus, in the present study, we investigated which P2Y2R-related signaling pathways are involved in the invasion of breast cancer cells. The highly metastatic breast cancer cells MDA-MB-231 and SK-BR-3 showed higher invasion than MCF-7 and T47D cells at a basal level, which was abolished through P2Y2R knockdown or in the presence of apyrase, an enzyme that hydrolyzes extracellular nucleotides. MDA-MB-231 cells also showed high levels of mesenchymal markers, such as Snail, Vimentin and N-cadherin, but not the epithelial marker E-cadherin and this expression was inhibited through ATP degradation or P2Y2R knockdown. Moreover, SK-BR-3 and MDA-MB231 cells exhibited higher ERK and PKC phosphorylation levels than T47D and MCF-7 cells and upregulated phospho-ERK and -PKC levels in MDA-MB-231 cells were significantly downregulated by apyrase or P2Y2R knockdown. Specific inhibitors of ERK, PKC and PLC markedly reduced the invasion and levels of mesenchymal marker expression in MDA-MB-231 cells. These results suggest that over-activated ERK and PKC pathways are involved in the P2Y2R-mediated invasion of breast cancer cells.

P2Y2 receptor activation by nucleotides released from highly metastatic breast cancer cells increases tumor growth and invasion via crosstalk with endothelial cells.

IntroductionExtracellular nucleotides are released and detectable in a high concentration within the tumor microenvironment. G protein-coupled P2Y2 nucleotide receptor (P2Y2R) is activated equipotently by adenosine triphosphate (ATP) and uridine 5′-triphosphate (UTP), which mediate proinflammatory responses such as cell migration and proliferation. However, the role of P2Y2R in the process of cancer metastasis remains unclear. This study aimed to determine the role of P2Y2R in the proliferation, migration and invasion of highly metastatic MDA-MB-231 breast cancer cells through crosstalk with endothelial cells (ECs).MethodsATP release and P2Y2R activity between high metastatic breast cancer cell MDA-MB-231 and low metastatic breast cancer cell MCF-7 were compared. Then, the role of P2Y2R on tumor growth and invasion via crosstalk with ECs was examined in vitro, using MDA-MB-231 cells and ECs transfected with control- or P2Y2R-siRNA, and in vivo, using an animal model injected with control-shRNA- or P2Y2R-shRNA-transfected MDA-MB-231 cells.ResultsWe found that this highly metastatic breast cancer cell line released higher levels of ATP and showed a higher P2Y2R activity in comparison to a low metastatic breast cancer cell line, MCF-7. In MDA-MB-231 cells, P2Y2R activation by ATP or UTP increased proliferation at 24 or 72 hours, which was abolished by P2Y2R knock-down. In addition, the adhesion of MDA-MB-231 cells to ECs and cell migration were both significantly increased by ATP or UTP through the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in MDA-MB-231 or ECs but not in cells where P2Y2R was knocked down. Furthermore, ATP- or UTP-mediated activation of P2Y2R induced MDA-MB-231 invasion through ECs, increased matrix metalloproteinase-9 (MMP-9) activity and vascular endothelial growth factor (VEGF) production in MDA-MB-231 and induced the phosphorylation of vascular endothelial (VE)-cadherin in ECs. Tumor growth and metastasis to other tissues were dramatically reduced, and body weight was increased in mice injected with P2Y2R-shRNA-transfected MDA-MB-231 cells compared to mice injected with control shRNA-transfected MDA-MB-231 cells.ConclusionThis study suggests that P2Y2R may play an important role in cancer metastasis via modulation of the crosstalk between cancer cells and ECs.Electronic supplementary materialThe online version of this article (doi:10.1186/bcr3694) contains supplementary material, which is available to authorized users.

Characterization of RNA in exosomes secreted by human breast cancer cell lines using next-generation sequencing

Exosomes are nanosized (30–100 nm) membrane vesicles secreted by most cell types. Exosomes have been found to contain various RNA species including miRNA, mRNA and long non-protein coding RNAs. A number of cancer cells produce elevated levels of exosomes. Because exosomes have been isolated from most body fluids they may provide a source for non-invasive cancer diagnostics. Transcriptome profiling that uses deep-sequencing technologies (RNA-Seq) offers enormous amount of data that can be used for biomarkers discovery, however, in case of exosomes this approach was applied only for the analysis of small RNAs. In this study, we utilized RNA-Seq technology to analyze RNAs present in microvesicles secreted by human breast cancer cell lines.Exosomes were isolated from the media conditioned by two human breast cancer cell lines, MDA-MB-231 and MDA-MB-436. Exosomal RNA was profiled using the Ion Torrent semiconductor chip-based technology. Exosomes were found to contain various classes of RNA with the major class represented by fragmented ribosomal RNA (rRNA), in particular 28S and 18S rRNA subunits. Analysis of exosomal RNA content revealed that it reflects RNA content of the donor cells. Although exosomes produced by the two cancer cell lines shared most of the RNA species, there was a number of non-coding transcripts unique to MDA-MB-231 and MDA-MB-436 cells. This suggests that RNA analysis might distinguish exosomes produced by low metastatic breast cancer cell line (MDA-MB-436) from that produced by highly metastatic breast cancer cell line (MDA-MB-231). The analysis of gene ontologies (GOs) associated with the most abundant transcripts present in exosomes revealed significant enrichment in genes encoding proteins involved in translation and rRNA and ncRNA processing. These GO terms indicate most expressed genes for both, cellular and exosomal RNA.For the first time, using RNA-seq, we examined the transcriptomes of exosomes secreted by human breast cancer cells. We found that most abundant exosomal RNA species are the fragments of 28S and 18S rRNA subunits. This limits the number of reads from other RNAs. To increase the number of detectable transcripts and improve the accuracy of their expression level the protocols allowing depletion of fragmented rRNA should be utilized in the future RNA-seq analyses on exosomes. Present data revealed that exosomal transcripts are representative of their cells of origin and thus could form basis for detection of tumor specific markers.

Abstract 998: Oncogenic potential of mitochondria regulate the cellular tumor characteristics

Abstract Mitochondrial-nucleus cross-talks and mitochondrial retrograde regulation of nuclear genes can play significant role in cellular properties. Several studies reported that somatic mitochondrial DNA alterations are often detected in cancer cells. Most of such alterations are polymorphisms without functional significance. Here we use a transmitochondrial cybrid (cybrids) approach to understand the significance of mitochondrial property in regulating the oncogenic properties of a cell irrespective of its mutational and nuclear status. Cybrid models in two differently defined nuclear backgrounds were studied. In the first model, mitochondria depleted rho zero (≥0) from an aggressive osteosarcoma cell lines 143B TK− was used as a common nuclear background. 143B β0 cells were fused with mitochondria from non-cancerous breast epithelial cell line MCF10A, low metastatic breast cancer cell line MDA-MB-468 and highly metastatic 143B TK− cell lines. In the second model, we used the common nuclear background of A1N4, a non-tumorigenic immortalized breast epithelial cell. A1N4 β0 cells were fused with MCF-10A and metastatic breast cancer cells MDA-MB-231. Studies in parental mitochondrial donor cells suggested that cancer cell lines have reduced ATP synthesis, oxygen consumption and respiratory chain activities compared to non-cancerous cells. Interestingly, in spite of the common nuclear backgrounds, cybrids also showed properties similar to their parental cells under both cancerous and non-cancerous nuclear backgrounds. Functional studies in cybrids under non-cancerous nuclear background observed that fusion with cancerous mitochondria resulted in loss of growth contact inhibition, induction of colony formation potential in soft agar and increased survival potential under serum starvation. Similarly, functional studies in cybrids with cancerous nuclear background suggested that non-cancerous mitochondria could reverse different oncogenic characteristics of cancer cell including cell proliferation, viability under hypoxic condition, anti-apoptotic properties, resistance to anti-cancer drug, invasion, colony formation in soft agar, and in vivo tumor growth in nude mice. Microarray analysis of cybrids under cancerous nuclear background suggested that several oncogenic pathways are inhibited due to non-cancerous mitochondria. Interestingly, under non-cancerous nuclear background, induction of important oncogenes was observed in cybrids containing cancerous mitochondria. These results suggest that mitochondrial-nuclear cross talk have significant contribution to cancer cell properties. Also it highlights mitochondria as a promising target in cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 998. doi:1538-7445.AM2012-998

Novel in vitro perfusion model to study the interaction between coagulation and blood‐borne metastasis

The association between cancer and hemostasis has long been studied in cell culture, animal models, and cancer patients developing thrombosis. The variety of biologic mechanisms involved in malignancy and metastasis makes the understanding of the relative importance of each mechanism difficult. We have developed a novel in vitro perfusion model that allows for the isolated study of the interactions between tumor cells and components of the hemostatic system under normal physiologic conditions. Segments of denuded umbilical cord or saphenous vein are cut longitudinally and mounted in a perfusion chamber under sterile conditions. Human breast cancer cells are perfused for 24 h under venous flow conditions with either whole blood (WB), platelet-rich plasma (PRP), platelet-poor plasma (PPP), or serum. Tissue samples are fixed and stained with hematoxylin and eosin as well as with pan-cytokeratin. Morphometric analysis is performed to quantify cancer cell adhesion. With PRP, this model maintains normal human physiologic conditions for the duration of the experiment. It differentiates between previously characterized high and low metastatic breast cancer cell lines. In addition, different vein tissue types do not alter tumor cell attachment. This model appears to be an accurate representation of the pathophysiology of in vivo metastasis. This model may serve as a useful bridge between cell culture studies and animal models. It may be a useful tool to elucidate the role of selected hemostatic systems in blood-borne metastasis and may potentially serve as a screening tool for the development of antimetastatic pharmaceutical agents.