H-Ras-specific upregulation of granulocyte colony-stimulating factor promotes human breast cell invasion via matrix metalloproteinase-2

Ras expression has been suggested to be a marker for tumor aggressiveness of breast cancer. We previously showed that H-Ras, but not N-Ras, induced invasive/migratory phenotypes in MCF10A human breast epithelial cells. The present study aimed to determine the role of granulocyte colony-stimulating factor in H-Ras-induced malignant progression of human breast epithelial cells. Here, we show that G-CSF plays a crucial role in H-Ras-induced MCF10A cell invasion and migration. The siRNA-mediated knockdown of G-CSF significantly reduced H-Ras-induced matrix metalloproteinase (MMP)-2 expression, as well as invasion/migration, suggesting the functional significance of G-CSF in the invasive phenotype of human breast cells. Importantly, the induction of G-CSF expression conferred the invasive/migratory phenotypes to MCF10A cells with up-regulation of MMP-2 and activation of Rac1, MKK3/6, p38 MAPK, Akt, and ERKs. Knockdown of Rac1 by siRNA significantly inhibited MMP-2 upregulation and invasiveness of G-CSF MCF10A cells, demonstrating that G-CSF-induced MMP-2 upregulation and invasive phenotype is mediated by Rac1. Using human breast tissues and sera from breast cancer patients, we further demonstrate that the expression level of G-CSF is strongly correlated with pathologically-diagnosed breast cancer. These data provide a molecular basis for the crucial role of G-CSF in promoting invasiveness of human breast epithelial cells.     

ATM Suppresses SATB1-Induced Malignant Progression in Breast Epithelial Cells

SATB1 drives metastasis when expressed in breast tumor cells by radically reprogramming gene expression. Here, we show that SATB1 also has an oncogenic activity to transform certain non-malignant breast epithelial cell lines. We studied the non-malignant MCF10A cell line, which is used widely in the literature. We obtained aliquots from two different sources (here we refer to them as MCF10A-1 and MCF10A-2), but found them to be surprisingly dissimilar in their responses to oncogenic activity of SATB1. Ectopic expression of SATB1 in MCF10A-1 induced tumor-like morphology in three-dimensional cultures, led to tumor formation in immunocompromised mice, and when injected into tail veins, led to lung metastasis. The number of metastases correlated positively with the level of SATB1 expression. In contrast, SATB1 expression in MCF10A-2 did not lead to any of these outcomes. Yet DNA copy-number analysis revealed that MCF10A-1 is indistinguishable genetically from MCF10A-2. However, gene expression profiling analysis revealed that these cell lines have significantly divergent signatures for the expression of genes involved in oncogenesis, including cell cycle regulation and signal transduction. Above all, the early DNA damage-response kinase, ATM, was greatly reduced in MCF10A-1 cells compared to MCF10A-2 cells. We found the reason for reduction to be phenotypic drift due to long-term cultivation of MCF10A. ATM knockdown in MCF10A-2 and two other non-malignant breast epithelial cell lines, 184A1 and 184B4, enabled SATB1 to induce malignant phenotypes similar to that observed for MCF10A-1. These data indicate a novel role for ATM as a suppressor of SATB1-induced malignancy in breast epithelial cells, but also raise a cautionary note that phenotypic drift could lead to dramatically different functional outcomes.     

Global gene expression profiling unveils S100A8/A9 as candidate markers in H-ras-mediated human breast epithelial cell invasion

The goal of the present study is to unveil the gene expression profile specific to the biological processes of human breast epithelial cell invasion and migration using an MCF10A model genetically engineered to constitutively activate the H-ras or N-ras signaling pathway. We previously showed that H-Ras, but not N-Ras, induces MCF10A cell invasion/migration, whereas both H-Ras and N-Ras induce cell proliferation and phenotypic transformation. Thus, these cell lines provide an experimental system to separate the gene expression profile associated with cell invasion apart from cell proliferation/transformation. Analysis of whole human genome microarray revealed that 412 genes were differentially expressed among MCF10A, N-Ras MCF10A, and H-Ras MCF10A cells and hierarchical clustering separated 412 genes into four clusters. We then tested whether S100A8 and S100A9, two of the genes which are most highly up-regulated in an H-Ras-specific manner, play a causative role for H-Ras-mediated MCF10A cell invasion and migration. Importantly, small interfering RNA-mediated knockdown of S100A8/A9 expression significantly reduced H-Ras-induced invasion/migration. Conversely, the induction of S100A8/A9 expression conferred the invasive/migratory phenotype to parental MCF10A cells. Furthermore, we provided evidence of signaling cross-talk between S100A8/A9 and the mitogen-activated protein kinase signaling pathways essential for H-Ras-mediated cell invasion and migration. Taken together, this study revealed S100A8/A9 genes as candidate markers for metastatic potential of breast epithelial cells. Our gene profile data provide useful information which may lead to the identification of additional potential targets for the prognosis and/or therapy of metastatic breast cancer.     

HuR Is Necessary for Mammary Epithelial Cell Proliferation and Polarity at Least in Part via ΔNp63

HuR, a RNA binding protein, is known to function as a tumor maintenance gene in breast cancer and associated with tumor growth and poor prognosis. However, the cellular function of this protein remains largely unknown in normal mammary epithelial cells. Here, we showed that in immortalized MCF10A mammary epithelial cells, HuR knockdown inhibits cell proliferation and enhances premature senescence. We also showed that in three-dimensional culture, MCF10A cells with HuR knockdown form abnormal acini with filled lumen and an aberrant expression pattern of the extracellular matrix protein laminin V. In addition, we showed that HuR knockdown increases ΔNp63, but decreases wild-type p53, expression in MCF10A cells. Moreover, we showed that ΔNp63 knockdown partially rescues the proliferative defect induced by HuR knockdown in MCF10A cells. Consistent with this, we identified two U-rich elements in the 3′-untranslated region of p63 mRNA, to which HuR specifically binds. Finally, we showed that HuR knockdown enhances ΔNp63 mRNA translation but has no effect on p63 mRNA turnover. Together, our data suggest that HuR maintains cell proliferation and polarity of mammary epithelial cells at least in part via ΔNp63.     

Hugl1 and Hugl2 in Mammary Epithelial Cells: Polarity,Proliferation, and Differentiation

Loss of epithelial polarity is described as a hallmark of epithelial cancer. To determine the role of Hugl1 and Hugl2 expression in the breast, we investigated their localization in human mammary duct tissue and the effects of expression modulation in normal and cancer cell lines on polarity, proliferation and differentiation. Expression of Hugl1 and Hugl2 was silenced in both MCF10A cells and Human Mammary Epithelial Cells and cell lines were grown in 2-D on plastic and in 3-D in Matrigel to form acini. Cells in monolayer were compared for proliferative and phenotypic changes while acini were examined for differences in size, ability to form a hollow lumen, nuclear size and shape, and localization of key domain-specific proteins as a measure of polarity. We detected overlapping but distinct localization of Hugl1 and Hugl2 in the human mammary gland, with Hugl1 expressed in both luminal and myoepithelium and Hugl2 largely restricted to myoepithelium. On a plastic surface, loss of Hugl1 or Hugl2 in normal epithelium induced a mesenchymal phenotype, and these cells formed large cellular masses when grown in Matrigel. In addition, loss of Hugl1 or Hugl2 expression in MCF10A cells resulted in increased proliferation on Matrigel, while gain of Hugl1 expression in tumor cells suppressed proliferation. Loss of polarity was also observed with knockdown of either Hugl1 or Hugl2, with cells growing in Matrigel appearing as a multilayered epithelium, with randomly oriented Golgi and multiple enlarged nuclei. Furthermore, Hugl1 knock down resulted in a loss of membrane identity and the development of cellular asymmetries in Human Mammary Epithelial Cells. Overall, these data demonstrate an essential role for both Hugl1 and Hugl2 in the maintenance of breast epithelial polarity and differentiated cell morphology, as well as growth control.     

Identification of CD63 as a tissue inhibitor of metalloproteinase-1 interacting cell surface protein

This study identified CD63, a member of the tetraspanin family, as a TIMP-1 interacting protein by yeast two-hybrid screening. Immunoprecipitation and confocal microscopic analysis confirmed CD63 interactions with TIMP-1, integrin beta1, and their co-localizations on the cell surface of human breast epithelial MCF10A cells. TIMP-1 expression correlated with the level of active integrin beta1 on the cell surface independent of cell adhesion. While MCF10A cells within a three-dimensional (3D) matrigel matrix form polarized acinar-like structures, TIMP-1 overexpression disrupted breast epithelial cell polarization and inhibited caspase-mediated apoptosis in centrally located cells, necessary for the formation and maintenance of the hollow acinar-like structures. Small hairpin RNA (shRNA)-mediated CD63 downregulation effectively reduced TIMP-1 binding to the cell surface, TIMP-1 co-localization with integrin beta1, and consequently reversed TIMP-1-mediated integrin beta1 activation, cell survival signaling and apoptosis inhibition. CD63 downregulation also restored polarization and apoptosis of TIMP-1 overexpressing MCF10A cells within a 3D-matrigel matrix. Taken together, the present study identified CD63 as a cell surface binding partner for TIMP-1, regulating cell survival and polarization via TIMP-1 modulation of tetraspanin/integrin signaling complex.     

Overexpression of MERTK Receptor Tyrosine Kinase in Epithelial Cancer Cells Drives Efferocytosis in a Gain-of-Function Capacity

MERTK, a member of the TAM (TYRO3, AXL, and MERTK) receptor tyrosine kinases, has complex and diverse roles in cell biology. On the one hand, knock-out of MERTK results in age-dependent autoimmunity characterized by failure of apoptotic cell clearance, while on the other, MERTK overexpression in cancer drives classical oncogene pathways leading to cell transformation. To better understand the interplay between cell transformation and efferocytosis, we stably expressed MERTK in human MCF10A cells, a non-tumorigenic breast epithelial cell line devoid of endogenous MERTK. While stable expression of MERTK in MCF10A resulted in enhanced motility and AKT-mediated chemoprotection, MERTK-10A cells did not form stable colonies in soft agar, or enhance proliferation compared with parental MCF10A cells. Concomitant to chemoresistance, MERTK also stimulated efferocytosis in a gain-of-function capacity. However, unlike AXL, MERTK activation was highly dependent on apoptotic cells, suggesting MERTK may preferentially interface with phosphatidylserine. Consistent with this idea, knockdown of MERTK in breast cancer cells MDA-MB 231 reduced efferocytosis, while transient or stable expression of MERTK stimulated apoptotic cell clearance in all cell lines tested. Moreover, human breast cancer cells with higher endogenous MERTK showed higher levels of efferocytosis that could be blocked by soluble TAM receptors. Finally, through MERTK, apoptotic cells induced PD-L1 expression, an immune checkpoint blockade, suggesting that cancer cells may adopt MERTK-driven efferocytosis as an immune suppression mechanism for their advantage. These data collectively identify MERTK as a significant link between cancer progression and efferocytosis, and a potentially unrealized tumor-promoting event when MERTK is overexpressed in epithelial cells.     

MiR-5047在乳腺癌细胞增殖迁移中的作用及表达调控*

探讨miR-5047在乳腺癌细胞中的表达及其在乳腺癌细胞增殖和迁移中的作用,并明确地西他滨在miR-5047表达调控中的作用。通过实时荧光定量PCR(qRT-PCR)检测人乳腺癌细胞系和正常乳腺上皮细胞MCF10A中miR-5047的表达水平;将miR-5047模拟物(mimic),阴性对照(NC)分别转染至MDA-MB-231和MCF7细胞,经平板克隆实验、MTT实验、划痕愈合实验检测乳腺癌细胞的增殖和迁移能力,通过qRT-PCR和Western blot检测相关基因表达及蛋白水平。使用浓度5 μmol/L和10 μmol/L的地西他滨分别处理MDA-MB-231和MCF-7细胞,经qRT-PCR检测不同浓度和处理时间条件下地西他滨对miR-5047表达的影响。同时,通过形态观察和Western blot检测地西他滨对乳腺癌细胞上皮间质转化的影响。与正常乳腺上皮细胞MCF-10A相比,miR-5047在乳腺癌细胞中表达均显著下调。miR-5047过表达可显著抑制乳腺癌细胞的增殖和迁移,促进上皮细胞标志物E-cadherin的表达,抑制间质细胞标志物Vimentin的表达。不同浓度地西他滨处理MDA-MB-231和MCF7细胞后,miR-5047表达均增强,且10 μmol/L作用48 h效果最显著。地西他滨可诱导MDA-MB-231细胞向上皮样转变。miR-5047在乳腺癌细胞系中表达显著下调,过表达miR-5047可抑制乳腺癌细胞的增殖和迁移,地西他滨可促进乳腺癌细胞中miR-5047的表达,并诱导细胞向上皮样转变。     

Decreased expression of EZH2 reactivates RASSF2A by reversal of promoter methylation in breast cancer cells

EZH2, the catalytic subunit of polycomb repressor complex 2, has oncogenic properties, whereas RASSF2A, a Ras association domain family protein, has a tumor suppressor role in many types of human cancer. However, the interrelationship between these two genes remains unclear. Here, we showed that the downregulation of EZH2 reduces CpG island methylation of the RASSF2A promoter, thereby leading to increased RASSF2A expression. Our findings also showed that knockdown of EZH2 increased RASSF2A expression in the human breast cancer cell line MCF‐7 in cooperation with DNMT1. This was similar to the effect of 5‐Aza‐CdR, a DNA methylation inhibitor that reactivates tumor suppressor genes and activated RASSF2A expression in our study. The EZH2 inhibitor DZNep markedly suppressed the proliferation, migration, and invasion of MCF‐7 cells treated with ADR and TAM. EZH2 inhibits the expression of tumor suppressor gene RASSF2A via promoter hypermethylation. Thus, it plays an important role in tumorigenesis and is a potential therapeutic target for the treatment of breast cancer.     

Characterization of morphological and cytoskeletal changes in MCF10A breast epithelial cells plated on laminin-5: comparison with breast cancer cell line MCF7

The extracellular matrix regulates functional and morphological differentiation of mammary epithelial cells both in vivo and in culture. The MCF10A human breast epithelial cell line is ideal for studying these processes because it retains many characteristics of normal breast epithelium. We describe a distinct set of morphological changes occurring in MCF10A cells plated on laminin-5, a component of the breast gland basement membrane extracellular matrix. MCF10A cells adhere and spread on laminin-5 about five times more rapidly than on fibronectin or uncoated surfaces. Within 10 minutes from plating on laminin-5, they send out microfilament-rich filopodia and by 30 minutes acquire a cobblestone appearance with microfilaments distributed around the cell periphery. At 90 minutes, with or without serum, > 75% of the MCF10A cells plated on laminin-5 remain in this stationary cobblestone phenotype, while the remainder takes on a motile appearance. Even after 18 hours, when the culture is likely entering an exponential growth phase, the majority of cells maintain a stationary cobblestone appearance, though motile cells have proportionally increased. In contrast, the fully transformed, malignant human breast epithelial cells, MCF7, never acquire a stationary cobblestone appearance, do not organize peripheral microfilaments, and throughout the early time points up to 120 min appear to be constantly motile on laminin-5. We propose that changes in morphology and microfilament organization in response to laminin-5 may represent a benchmark for distinguishing normal vs. malignant behavior of epithelial cells derived from the mammary gland. This may lead to better model systems for studying the interactions between breast epithelium and the basement membrane extracellular matrix, which appear to be deregulated in processes like carcinogenesis and metastasis.     

Characterization of Morphological and Cytoskeletal Changes in MCF10A Breast Epithelial Cells Plated on Laminin-5: Comparison with Breast Cancer Cell Line MCF7

The extracellular matrix regulates functional and morphological differentiation of mammary epithelial cells both in vivo and in culture. The MCF10A human breast epithelial cell line is ideal for studying these processes because it retains many characteristics of normal breast epithelium. We describe a distinct set of morphological changes occurring in MCF10A cells plated on laminin-5, a component of the breast gland basement membrane extracellular matrix. MCF10A cells adhere and spread on laminin-5 about five times more rapidly than on fibronectin or uncoated surfaces. Within 10 minutes from plating on laminin-5, they send out microfilament-rich filopodia and by 30 minutes acquire a cobblestone appearance with microfilaments distributed around the cell periphery. At 90 minutes, with or without serum, >75% of the MCF10A cells plated on laminin-5 remain in this stationary cobblestone phenotype, while the remainder takes on a motile appearance. Even after 18 hours, when the culture is likely entering an exponential growth phase, the majority of cells maintain a stationary cobblestone appearance, though motile cells have proportionally increased. In contrast, the fully transformed, malignant human breast epithelial cells, MCF7, never acquire a stationary cobblestone appearance, do not organize peripheral microfilaments, and throughout the early time points up to 120 min appear to be constantly motile on laminin-5. We propose that changes in morphology and microfilament organization in response to laminin-5 may represent a benchmark for distinguishing normal vs. malignant behavior of epithelial cells derived from the mammary gland. This may lead to better model systems for studying the interactions between breast epithelium and the basement membrane extracellular matrix, which appear to be deregulated in processes like carcinogenesis and metastasis.     

LncRNA AC009686.2促进乳腺癌细胞增殖和侵袭

长非编码RNAs(long non-coding RNAs,LncRNAs)作为一类基因表达的调控因子,在多种肿瘤的发生发展中发挥关键作用,然而LncRNAs在乳腺癌中的作用及相关机制尚未完全阐明.为了寻找在乳腺癌发生发展中起关键作用的LncRNAs,本研究通过分析TCGA数据库发现,LncRNA AC009686.2...     

Nuclear factor-kappaB accounts for the repressor effects of high stromal cell-derived factor-1alpha levels on Tac1 expression in nontumorigenic breast cells

Stromal cell-derived factor-1alpha (SDF-1alpha) is a CXC chemokine that interacts with CXCR4 receptor. Tac1 encodes peptides belonging to the tachykinins, including substance P. SDF-1alpha production is decreased in Tac1 knockdown breast cancer cells and is also reduced in these cancer cells following contact with bone marrow stroma when Tac1 expression is increased. Here, we report on the effects of relatively high and low SDF-1alpha levels on Tac1 expression in nontumorigenic breast cells MCF12A. Reporter gene assays, Northern analyses, and ELISA for substance P showed increased Tac1 expression at 20 and 50 ng/mL SDF-1alpha and reduced expression at 100 ng/mL. Omission of the untranslated region showed a dose-dependent effect of SDF-1alpha on reporter gene activity, suggesting that receptor desensitization cannot account for the suppressive effects at 100 ng/mL SDF-1alpha. Tac1 expression at high SDF-1alpha involves an intracellular signaling pathway that incorporates the activation of phosphatidylinositol 3-kinase-phosphoinositide-dependent kinase-1-AKT-nuclear factor-kappaB (NF-kappaB). The major repressive effect occurs via NF-kappaB located within exon 1. In summary, NF-kappaB is involved in the repression of Tac1 at higher levels of SDF-1alpha in MCF12A. These results are relevant to dysfunction of Tac1 in breast cancer cells and also provide insights on the behavior of breast cancer cells as they traverse across gradient changes of SDF-1alpha.     

Acetonic extract of Buxus sempervirens induces cell cycle arrest, apoptosis and autophagy in breast cancer cells

Plants are an invaluable source of potential new anti-cancer drugs. Here, we investigated the cytotoxic activity of the acetonic extract of Buxus sempervirens on five breast cancer cell lines, MCF7, MCF10CA1a and T47D, three aggressive triple positive breast cancer cell lines, and BT-20 and MDA-MB-435, which are triple negative breast cancer cell lines. As a control, MCF10A, a spontaneously immortalized but non-tumoral cell line has been used. The acetonic extract of Buxus sempervirens showed cytotoxic activity towards all the five studied breast cancer cell lines with an IC(50) ranging from 7.74 μg/ml to 12.5 μg/ml. Most importantly, the plant extract was less toxic towards MCF10A with an IC(50) of 19.24 μg/ml. Fluorescence-activated cell sorting (FACS) analysis showed that the plant extract induced cell death and cell cycle arrest in G0/G1 phase in MCF7, T47D, MCF10CA1a and BT-20 cell lines, concomitant to cyclin D1 downregulation. Application of MCF7 and MCF10CA1a respective IC(50) did not show such effects on the control cell line MCF10A. Propidium iodide/Annexin V double staining revealed a pre-apoptotic cell population with extract-treated MCF10CA1a, T47D and BT-20 cells. Transmission electron microscopy analyses indicated the occurrence of autophagy in MCF7 and MCF10CA1a cell lines. Immunofluorescence and Western blot assays confirmed the processing of microtubule-associated protein LC3 in the treated cancer cells. Moreover, we have demonstrated the upregulation of Beclin-1 in these cell lines and downregulation of Survivin and p21. Also, Caspase-3 detection in treated BT-20 and T47D confirmed the occurrence of apoptosis in these cells. Our findings indicate that Buxus sempervirens extract exhibit promising anti-cancer activity by triggering both autophagic cell death and apoptosis, suggesting that this plant may contain potential anti-cancer agents for single or combinatory cancer therapy against breast cancer.     

Tumor cells induce the cancer associated fibroblast phenotype via caveolin-1 degradation: Implications for breast cancer and DCIS therapy with autophagy inhibitors

Loss of stromal caveolin 1 (Cav-1) is a novel biomarker for cancer-associated fibroblasts that predicts poor clinical outcome in breast cancer and DCIS patients. We hypothesized that epithelial cancer cells may have the ability to drive Cav-1 downregulation in adjacent normal fibroblasts, thereby promoting the cancer associated fibroblast phenotype. To test this hypothesis directly, here we developed a novel co-culture model employing (i) human breast cancer cells (MCF7), and (ii) immortalized fibroblasts (hTERT-BJ1), which are grown under defined experimental conditions. Importantly, we show that co-culture of immortalized human fibroblasts with MCF7 breast cancer cells leads to Cav-1 downregulation in fibroblasts. These results were also validated using primary cultures of normal human mammary fibroblasts co-cultured with MCF7 cells. In this system, we show that Cav-1 downregulation is mediated by autophagic/lysosomal degradation, as pre-treatment with lysosome-specific inhibitors rescues Cav-1 expression. Functionally, we demonstrate that fibroblasts co-cultured with MCF7 breast cancer cells acquire a cancer associated fibroblast phenotype, characterized by Cav-1 downregulation, increased expression of myofibroblast markers and extracellular matrix proteins, and constitutive activation of TGFβ/Smad2 signaling. siRNA-mediated Cav-1 downregulation mimics several key changes that occur in co-cultured fibroblasts, clearly indicating that a loss of Cav-1 is a critical initiating factor, driving stromal fibroblast activation during tumorigenesis. As such, this co-culture system can now be used as an experimental model for generating “synthetic” cancer associated fibroblasts (CAFs). More specifically, these “synthetic” CAFs could be used for drug screening to identify novel therapeutics that selectively target the Cav-1-negative tumor micro-environment. Our findings also suggest that chloroquine, or other autophagy/lysosome inhibitors, may be useful as anti-cancer agents, to therapeutically restore the expression of stromal Cav-1 in cancer associated fibroblasts. We discuss this possibility, in light of the launch of a new clinical trial that uses chloroquine to treat DCIS patients: PINC (Preventing Invasive Breast Neoplasia with Cholorquine) [See http://clinicaltrials.gov/show/NCT01023477].     

Centchroman suppresses breast cancer metastasis by reversing epithelial–mesenchymal transition via downregulation of HER2/ERK1/2/MMP-9 signaling

Metastatic spread during carcinogenesis worsens disease prognosis and accelerates the cancer progression. Therefore, newer therapeutic options with higher specificity toward metastatic cancer are required. Centchroman (CC), a female oral contraceptive, has previously been reported to possess antiproliferative and proapoptotic activities in human breast cancer cells. Here, we investigated the effect of CC-treatment against breast cancer metastasis and associated molecular mechanism using in vitro and in vivo models. CC significantly inhibited the proliferation of human and mouse mammary cancer cells. CC-treatment also inhibited migration and invasion capacities of highly metastatic MDA-MB-231 and 4T1 cells, at sub-IC₅₀ concentrations. Inhibition of cell migration and invasion was found to be associated with the reversal of epithelial-to-mesenchymal transition (EMT) as observed by the upregulation of epithelial markers and downregulation of mesenchymal markers as well as decreased activities of matrix metalloproteinases. Experimental EMT induced by exposure to TGFβ/TNFα in nontumorigenic human mammary epithelial MCF10A cells was also reversed by CC as evidenced by morphological changes and modulation in the expression levels of EMT-markers. CC-mediated inhibition of cellular migration was, at least partially, mediated through inhibition of ERK1/2 signaling, which was further validated by using MEK1/2 inhibitor (PD0325901). Furthermore, CC-treatment resulted in suppression of tumor growth and lung metastasis in 4T1-syngeneic mouse model. Collectively, our findings suggest that CC-treatment at higher doses specifically induces cellular apoptosis and inhibits cellular proliferation; whereas at lower doses, it inhibits cellular migration and invasion. Therefore, CC could further be developed as an effective drug candidate against metastatic breast cancer.