Anti-cancer activities of tea epigallocatechin-3-gallate in breast cancer patients under radiotherapy

The purpose of this study was to test the hypothesis that administration of epigallocatechin-3-gallate (EGCG), a polyphenol present in abundance in widely consumed tea, inhibits cell proliferation, invasion, and angiogenesis in breast cancer patients. EGCG in 400 mg capsules was orally administered three times daily to breast cancer patients undergoing treatment with radiotherapy. Parameters related to cell proliferation, invasion, and angiogenesis were analyzed while blood samples were collected at different time points to determine efficacy of the EGCG treatment. Compared to patients who received radiotherapy alone, those given radiotherapy plus EGCG for an extended time period (two to eight weeks) showed significantly lower serum levels of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and reduced activation of metalloproteinase-9 and metalloproteinase-2 (MMP9/MMP2). Addition of sera obtained from patients treated with combination of radiotherapy and EGCG feeding for 2-8 weeks to in vitro cultures of highly-metastatic human MDA-MB-231 breast cancer cells resulted in the following significant changes: (1) suppression of cell proliferation and invasion; (2) arrest of cell cycles at the G0/G1 phase; (3) reduction of activation of MMP9/MMP2, expressions of Bcl-2/Bax, c-Met receptor, NF-κB, and the phosphorylation of Akt. MDA-MB-231 cells exposed to 5-10 μM EGCG also showed significant augmentation of the apoptosis inducing effects of γ-radiation, concomitant with reduced NF-κB protein level and AKT phosphorylation. These results provide hitherto unreported evidence that EGCG potentiated efficacy of radiotherapy in breast cancer patients, and raise the possibility that this tea polyphenol has potential to be a therapeutic adjuvant against human metastatic breast cancer.     

Anandamide inhibits adhesion and migration of breast cancer cells

The endocannabinoid system regulates cell proliferation in human breast cancer cells. We reasoned that stimulation of cannabinoid CB1 receptors could induce a non-invasive phenotype in breast metastatic cells. In a model of metastatic spreading in vivo, the metabolically stable anandamide analogue, 2-methyl-2'-F-anandamide (Met-F-AEA), significantly reduced the number and dimension of metastatic nodes, this effect being antagonized by the selective CB1 antagonist SR141716A. In MDA-MB-231 cells, a highly invasive human breast cancer cell line, and in TSA-E1 cells, a murine breast cancer cell line, Met-F-AEA inhibited adhesion and migration on type IV collagen in vitro without modifying integrin expression: both these effects were antagonized by SR141716A. In order to understand the molecular mechanism involved in these processes, we analyzed the phosphorylation of FAK and Src, two tyrosine kinases involved in migration and adhesion. In Met-F-AEA-treated cells, we observed a decreased tyrosine phosphorylation of both FAK and Src, this effect being attenuated by SR141716A. We propose that CB1 receptor agonists inhibit tumor cell invasion and metastasis by modulating FAK phosphorylation, and that CB1 receptor activation might represent a novel therapeutic strategy to slow down the growth of breast carcinoma and to inhibit its metastatic diffusion in vivo.     

CXCR4/CXCL12 participate in extravasation of metastasizing breast cancer cells within the liver in a rat model

Introduction

Organ-specific composition of extracellular matrix proteins (ECM) is a determinant of metastatic host organ involvement. The chemokine CXCL12 and its receptor CXCR4 play important roles in the colonization of human breast cancer cells to their metastatic target organs. In this study, we investigated the effects of chemokine stimulation on adhesion and migration of different human breast cancer cell lines in vivo and in vitro with particular focus on the liver as a major metastatic site in breast cancer.

Methods

Time lapse microscopy, in vitro adhesion and migration assays were performed under CXCL12 stimulation. Activation of small GTPases showed chemokine receptor signalling dependence from ECM components. The initial events of hepatic colonisation of MDA-MB-231 and MDA-MB-468 cells were investigated by intravital microscopy of the liver in a rat model and under shRNA inhibition of CXCR4.

Results

In vitro, stimulation with CXCL12 induced increased chemotactic cell motility (p<0.05). This effect was dependent on adhesive substrates (type I collagen, fibronectin and laminin) and induced different responses in small GTPases, such as RhoA and Rac-1 activation, and changes in cell morphology. In addition, binding to various ECM components caused redistribution of chemokine receptors at tumour cell surfaces. In vivo, blocking CXCR4 decreased extravasation of highly metastatic MDA-MB-231 cells (p<0.05), but initial cell adhesion within the liver sinusoids was not affected. In contrast, the less metastatic MDA-MB-468 cells showed reduced cell adhesion but similar migration within the hepatic microcirculation. Conclusion: Chemokine-induced extravasation of breast cancer cells along specific ECM components appears to be an important regulator but not a rate-limiting factor of their metastatic organ colonization.     

Mesenchymal stem cell-derived interleukin-6 and vascular endothelial growth factor promote breast cancer cell migration

Several different cytokines and growth factors secreted by mesenchymal stem cells (MSCs) have been hypothesized to play a role in breast cancer progression. By using a small panel of breast cancer cell lines (MCF‐7, T47D, and SK‐Br‐3 cells), we analyzed the role of interleukin‐6 (IL‐6) and vascular endothelial growth factor A (VEGF) in the cross‐talk between MSCs and breast cancer cells. We performed migration assays in which breast cancer cells were allowed to migrate in response to conditioned medium from MSCs (MSCs‐CM), in absence or in presence of the anti‐VEGF antibody bevacizumab or an anti‐IL‐6 antibody, alone or in combination. We found that anti‐VEGF and anti‐IL‐6 antibodies inhibited the migration of breast cancer cells and that the combination had an higher inhibitory effect. We next evaluated the effects of recombinant VEGF and IL‐6 proteins on breast cancer cell growth and migration. IL‐6 and VEGF had not significant effects on the proliferation of breast carcinoma cells. In contrast, both VEGF and IL‐6 significantly increased the ability to migrate of MCF‐7, T47D and SK‐Br‐3 cells, with the combination showing a greater effect as compared with treatment with a single protein. The combination of VEGF and IL‐6 produced in breast cancer cells a more significant and more persistent activation of MAPK, AKT, and p38MAPK intracellular signaling pathways. These results suggest that MSC‐secreted IL‐6 and VEGF may act as paracrine factors to sustain breast cancer cell migration. J. Cell. Biochem. 113: 3363–3370, 2012. © 2012 Wiley Periodicals, Inc.     

Gene-engineered T cells as a superior adjuvant therapy for metastatic cancer

The major limiting factor in the successful application of adjuvant therapy for metastatic disease is the lack of adjuvant specificity that leads to severe side effects. Reasoning that T cells of the immune system are highly specific, we generated tumor-specific T cells by genetic modification of mouse primary T cells with a chimeric receptor reactive with the human breast cancer-associated Ag erbB-2. These T cells killed breast cancer cells and secreted IFN-gamma in an Ag-specific manner in vitro. We investigated their use against metastatic breast cancer in mice in an adjuvant setting, and compared their effectiveness with the commonly applied adjuvants doxorubicin, 5-fluorouracil, and herceptin. Mice were inoculated orthotopically with the human erbB-2-expressing spontaneously metastatic mouse breast cancer 4T1.2 in mammary tissue, and the primary tumor was surgically removed 8 days later. Significant metastatic disease was demonstrated in lung and liver at the time of surgery on day 8 with increased tumor burden at later time points. T cell adjuvant treatment of day 8 metastatic disease resulted in dramatic increases in survival of mice, and this survival was significantly greater than that afforded by either doxorubicin, 5-fluorouracil, or herceptin.     

Vascular endothelial growth factor modulates the transendothelial migration of MDA-MB-231 breast cancer cells through regulation of brain microvascular endothelial cell permeability

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), has been shown to increase potently the permeability of endothelium and is highly expressed in breast cancer cells. In this study, we investigated the role of VEGF/VPF in breast cancer metastasis to the brain. Very little is known about the role of endothelial integrity in the extravasation of breast cancer cells to the brain. We hypothesized that VEGF/VPF, having potent vascular permeability activity, may support tumor cell penetration across blood vessels by inducing vascular leakage. To examine this role of VEGF/VPF, we used a Transwell culture system of the human brain microvascular endothelial cell (HBMEC) monolayer as an in vitro model for the blood vessels. We observed that VEGF/VPF significantly increased the penetration of the highly metastatic MDA-MB-231 breast cancer cells across the HBMEC monolayer. We found that the increased transendothelial migration (TM) of MDA-MB-231 cells resulted from the increased adhesion of tumor cells onto the HBMEC monolayer. These effects (TM and adhesion of tumor cells) were inhibited by the pre-treatment of the HBMEC monolayer with the VEGF/VPF receptor (KDR/Flk-1) inhibitor, SU-1498, and the calcium chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl)ester. These treatments of the HBMEC monolayer also inhibited VEGF/VPF-induced permeability and the cytoskeletal rearrangement of the monolayer. These data suggest that VEGF/VPF can modulate the TM of tumor cells by regulating the integrity of the HBMEC monolayer. Taken together, these findings indicate that VEGF/VPF might contribute to breast cancer metastasis by enhancing the TM of tumor cells through the down-regulation of endothelial integrity.     

MDA-MB-435 human breast carcinoma cell homo- and heterotypic adhesion under flow conditions is mediated in part by Thomsen-Friedenreich antigen-galectin-3 interactions

The importance of Thomsen-Friedenreich antigen (T antigen)-galectin-3 interactions in adhesion of human breast carcinoma cells to the endothelium under conditions of flow was studied. Highly metastatic cells (MDA-MB-435) expressing high levels of both galectin-3 and T antigen demonstrated significantly increased adhesion to monolayers of endothelial cells compared with their non-metastatic counterpart (MDA-MB-468) in vitro. Within minutes of adhesion, the highly metastatic cells acquire the ability of enhanced homotypic adhesion, leading to the formation of multicellular aggregates at sites of attachment to endothelial cells in vitro. Treatment of cells with lactulosyl-l-leucine, a synthetic T antigen antagonist that targets galectin-3 by mimicking T antigen, caused a 60-80% inhibition of both homo- and heterotypic adhesion of MDA-MB-435 cells. Confocal microscopy and fluorescence-activated cell sorter analysis revealed redistribution of endothelial galectin-3 to the site of heterotypic intercellular contacts, whereas galectin-3 in MDA-MB-435 cells accumulated at sites of homotypic interaction. MDA-MB-435 cells also exhibited increased adhesion and intravascular retention within the microvessels of transplanted lung allografts in nude mice. T antigen and galectin-3-mediated interactions of metastatic cancer cells with endothelium under conditions of flow are characterized by a unique adhesion mechanism that qualitatively distinguishes their homo- and heterotypic adhesive behavior from other cell types such as leukocytes.     

Angiotensin II facilitates breast cancer cell migration and metastasis

Breast cancer metastasis is a leading cause of death by malignancy in women worldwide. Efforts are being made to further characterize the rate-limiting steps of cancer metastasis, i.e. extravasation of circulating tumor cells and colonization of secondary organs. In this study, we investigated whether angiotensin II, a major vasoactive peptide both produced locally and released in the bloodstream, may trigger activating signals that contribute to cancer cell extravasation and metastasis. We used an experimental in vivo model of cancer metastasis in which bioluminescent breast tumor cells (D3H2LN) were injected intra-cardiacally into nude mice in order to recapitulate the late and essential steps of metastatic dissemination. Real-time intravital imaging studies revealed that angiotensin II accelerates the formation of metastatic foci at secondary sites. Pre-treatment of cancer cells with the peptide increases the number of mice with metastases, as well as the number and size of metastases per mouse. In vitro, angiotensin II contributes to each sequential step of cancer metastasis by promoting cancer cell adhesion to endothelial cells, trans-endothelial migration and tumor cell migration across extracellular matrix. At the molecular level, a total of 102 genes differentially expressed following angiotensin II pre-treatment were identified by comparative DNA microarray. Angiotensin II regulates two groups of connected genes related to its precursor angiotensinogen. Among those, up-regulated MMP2/MMP9 and ICAM1 stand at the crossroad of a network of genes involved in cell adhesion, migration and invasion. Our data suggest that targeting angiotensin II production or action may represent a valuable therapeutic option to prevent metastatic progression of invasive breast tumors.     

Osteoblasts are a major source of inflammatory cytokines in the tumor microenvironment of bone metastatic breast cancer

Metastatic breast cancer cells co‐opt the cells of the bone to increase their production of inflammatory cytokines. Here, we sought to identify key cytokines expressed by osteoblasts in vitro and in vivo in the presence of MDA‐MB‐231 metastatic breast cancer cells, including a bone‐seeking variant. We hypothesized that osteoblast‐derived cytokines increase in the presence of metastatic breast cancer cell conditioned medium (CM), act as chemoattractants for cancer cells, and enhance osteoclast formation. We detected increases in the concentrations of osteoblast‐derived IL‐6, MCP‐1, VEGF, MIP‐2, and KC in vitro in culture supernatants from MC3T3‐E1 cells in the presence of metastatic breast cancer cell CM and from cancer‐bearing femurs ex vivo. A comparison of cancer cell‐ and osteoblast‐derived cytokines revealed that while breast cancer cells expressed the same or equivalent cytokines as the osteoblasts, the breast cancer cells only produced picogram quantities of MCP‐1; osteoblasts expressed nanogram amounts. Bone‐derived MCP‐1 increased in the proximal metaphysis, an area where breast cancer cells preferentially trafficked following intracardiac inoculation in athymic mice. An MDA‐MB‐231 bone‐seeking variant was not different from parental lines. Osteoblast CM was a potent chemoattractant for metastatic breast cancer cells. Furthermore, culture supernatants of osteoblasts treated with breast cancer cell CM enhanced osteoclast formation. These findings suggest that bone metastatic breast cancer cells utilize osteoblast‐derived cytokines to facilitate breast cancer cell colonization and survival upon arrival in the bone microenvironment. J. Cell. Biochem. 111: 1138–1148, 2010. © 2010 Wiley‐Liss, Inc.     

A novel metformin derivative,HL010183, inhibits proliferation and invasion of triple-negative breast cancer cells

Mounting evidence suggests that metformin (N,N-dimethylbiguanide), a widely prescribed drug for the treatment of type II diabetes, exerts an anti-tumor effect on several cancers including breast cancer. Breast cancer has been estimated as one of the most commonly diagnosed types of cancer among women. In particular, triple-negative breast cancers are associated with poor prognosis and metastatic growth. In the present study, we synthesized a novel metformin derivative 5 (HL010183) and metformin salts, 9a, 9b, and 9c (metformin gamma-aminobutyric acid (GABA) salt, metformin pregabalin salt and metformin gabapentin salt), which exerted more potent inhibitory effects on the proliferation and invasiveness of Hs578T triple-negative breast carcinoma cells than metformin. Importantly, 5 showed approximately 100-fold more potent effects compared to metformin. In a triple-negative breast cancer xenograft model, 5 showed a comparable degree of inhibitory effect on in vivo tumor growth at the 100 mg/kg dose to that of metformin at 500 mg/kg. Our results clearly demonstrate that 5 exerts a potent anti-tumor effect both in vitro and in vivo, paving the way for a strategy for treatment of triple-negative breast cancer.     

Metformin induces unique biological and molecular responses in triple negative breast cancer cells

Triple negative (TN) breast cancer is more frequent in women who are obese or have type II diabetes, as well as young Women of Color. These cancers do not express receptors for the steroid hormones estrogen or progesterone, or the type II receptor tyrosine kinase (RTK) Her-2 but do have upregulation of basal cytokeratins and the epidermal growth factor (EGFR). These data suggest that aberrations of glucose and fatty acid metabolism, signaling through EGFR and genetic factors may promote the development of TN cancers. The anti-type II diabetes drug metformin has been associated with a decreased incidence of breast cancer, although the specific molecular subtypes that may be reduced by metformin have not been reported. Our data indicates that metformin has unique anti-TN breast cancer effects both in vitro and in vivo. It inhibits cell proliferation (with partial S phase arrest), colony formation and induces apoptosis via activation of the intrinsic and extrinsic signaling pathways only in TN breast cancer cell lines. At the molecular level, metformin increases P-AMPK, reduces P-EGFR, EGFR, P-MAPK, P-Src, cyclin D1 and cyclin E (but not cyclin A or B, p27 or p21), and induces PARP cleavage in a dose- and time-dependent manner. These data are in stark contrast to our previously published biological and molecular effects of metformin on luminal A and B, or Her-2 type breast cancer cells. Nude mice bearing tumor xenografts of the TN line MDA-MB-231, treated with metformin, show significant reductions in tumor growth (p=0.0066) and cell proliferation (p=0.0021) as compared to untreated controls. Metformin pre-treatment, before injection of MDA-MB-231 cells, results in a significant decrease in tumor outgrowth and latency. Given the unique anti-cancer activity of metformin against TN disease, both in vitro and in vivo, it should be explored as a therapeutic agent against this aggressive form of breast cancer.     

Cancer Associated Fibroblasts Promote Tumor Growth and Metastasis by Modulating the Tumor Immune Microenvironment in a 4T1 Murine Breast Cancer Model

Background

Local inflammation associated with solid tumors commonly results from factors released by tumor cells and the tumor stroma, and promotes tumor progression. Cancer associated fibroblasts comprise a majority of the cells found in tumor stroma and are appealing targets for cancer therapy. Here, our aim was to determine the efficacy of targeting cancer associated fibroblasts for the treatment of metastatic breast cancer.

Methodology/Principal Findings

We demonstrate that cancer associated fibroblasts are key modulators of immune polarization in the tumor microenvironment of a 4T1 murine model of metastatic breast cancer. Elimination of cancer associated fibroblasts in vivo by a DNA vaccine targeted to fibroblast activation protein results in a shift of the immune microenvironment from a Th2 to Th1 polarization. This shift is characterized by increased protein expression of IL-2 and IL-7, suppressed recruitment of tumor-associated macrophages, myeloid derived suppressor cells, T regulatory cells, and decreased tumor angiogenesis and lymphangiogenesis. Additionally, the vaccine improved anti-metastatic effects of doxorubicin chemotherapy and enhanced suppression of IL-6 and IL-4 protein expression while increasing recruitment of dendritic cells and CD8⁺ T cells. Treatment with the combination therapy also reduced tumor-associated Vegf, Pdgfc, and GM-CSF mRNA and protein expression.

Conclusions/Significance

Our findings demonstrate that cancer associated fibroblasts promote tumor growth and metastasis through their role as key modulators of immune polarization in the tumor microenvironment and are valid targets for therapy of metastatic breast cancer.     

Matrilysin (MMP‐7) cleaves C‐type lectin domain family 3 member A (CLEC3A) on tumor cell surface and modulates its cell adhesion activity

Matrilysin (MMP‐7) plays important roles in tumor progression. Previous studies have suggested that MMP‐7 binds to tumor cell surface and promotes their metastatic potential. In this study, we identified C‐type lectin domain family 3 member A (CLEC3A) as a membrane‐bound substrate of MMP‐7. Although this protein is known to be expressed specifically in cartilage, its message was found in normal breast and breast cancer tissues as well as breast and colon cancer cell lines. Because few studies have been done on CLEC3A, we overexpressed its recombinant protein in human cancer cells. CLEC3A was found in the cell membrane, extracellular matrix (ECM), and culture medium of the CLEC3A‐expressing cells. CLEC3A has a basic sequence in the NH₂‐terminal domain and showed a strong heparin‐binding activity. MMP‐7 cleaved the 20‐kDa CLEC3A protein, dividing it to a 15‐kDa COOH‐terminal fragment and an NH₂‐terminal fragment with the basic sequence. The 15‐kDa fragment no longer had heparin‐binding activity. Treatment of the CLEC3A‐expressing cells with MMP‐7 released the 15‐kDa CLEC3A into the culture supernatant. Furthermore, the 20‐kDa CLEC3A promoted cell adhesion to laminin‐332 and fibronectin substrates, but this activity was abrogated by the cleavage by MMP‐7. These results suggest that CLEC3A binds to heparan sulfate proteoglycans on cell surface, leading to the enhancement of cell adhesion to integrin ligands on ECM. It can be speculated that the cleavage of CLEC3A by MMP‐7 weakens the stable adhesion of tumor cells to the matrix and promotes their migration in tumor microenvironments. J. Cell. Biochem. 106: 693–702, 2009. © 2009 Wiley‐Liss, Inc.     

Activated T cell exosomes promote tumor invasion via Fas signaling pathway

Activated T cells release bioactive Fas ligand (FasL) in exosomes, which subsequently induce self-apoptosis of T cells. However, their potential effects on cell apoptosis in tumors are still unknown. In this study, we purified exosomes expressing FasL from activated CD8(+) T cell from OT-I mice and found that activated T cell exosomes had little effect on apoptosis and proliferation of tumor cells but promoted the invasion of B16 and 3LL cancer cells in vitro via the Fas/FasL pathway. Activated T cell exosomes increased the amount of cellular FLICE inhibitory proteins and subsequently activated the ERK and NF-κB pathways, which subsequently increased MMP9 expression in the B16 murine melanoma cells. In a tumor-invasive model in vivo, we observed that the activated T cell exosomes promoted the migration of B16 tumor cells to lung. Interestingly, pretreatment with FasL mAb significantly reduced the migration of B16 tumor cells to lung. Furthermore, CD8 and FasL double-positive exosomes from tumor mice, but not normal mice, also increased the expression of MMP9 and promoted the invasive ability of B16 murine melanoma and 3LL lung cancer cells. In conclusion, our results indicate that activated T cell exosomes promote melanoma and lung cancer cell metastasis by increasing the expression of MMP9 via Fas signaling, revealing a new mechanism of tumor immune escape.     

Combined resveratrol, quercetin, and catechin treatment reduces breast tumor growth in a nude mouse model

Grape polyphenols can act as antioxidants, antiangiogenics, and selective estrogen receptor (ER) modifiers and are therefore especially relevant for gynecological cancers such as breast cancer. The major polyphenols of red wine (resveratrol, quercetin, and catechin) have been individually shown to have anticancer properties. However, their combinatorial effect on metastatic breast cancers has not been investigated in vivo. We tested the effect of low dietary concentrations of resveratrol, quercetin, and catechin on breast cancer progression in vitro by analyzing cell proliferation and cell cycle progression. The effects of these compounds on fluorescently tagged breast tumor growth in nude mice were assessed using in situ fluorescence image analysis. Individual polyphenols at 0.5 microM neither decreased breast cancer cell proliferation nor affected cell cycle progression in vitro. However, a combination of resveratrol, quercetin, and catechin at 0.5, 5, or 20 microM each significantly reduced cell proliferation and blocked cell cycle progression in vitro. Furthermore, using in situ image analysis, we determined that combined dietary polyphenols at 0.5, 5, or 25 mg/kg reduced primary tumor growth of breast cancer xenografts in a nude mouse model. Peak inhibition was observed at 5 mg/kg. These results indicate that grape polyphenols may inhibit breast cancer progression.