Metastatic 4T1 Breast Cancer Research Articles

Antitumor effect and toxicity of free rhodium (II) citrate and rhodium (II) citrate-loaded maghemite nanoparticles in mice bearing breast cancer.

BackgroundMagnetic fluids containing superparamagnetic iron oxide nanoparticles represent an attractive platform as nanocarriers in chemotherapy. Recently, we developed a formulation of maghemite nanoparticles coated with rhodium (II) citrate, which resulted in in vitro cytotoxicity enhanced up to 4.6 times when compared to free rhodium (II) citrate formulation on breast carcinoma cells. In this work, we evaluate the antitumor activity and toxicity induced by these formulations in Balb/c mice bearing orthotopic 4T1 breast carcinoma.MethodsMice were evaluated with regard to the treatments’ toxicity through analyses of hemogram, serum levels of alanine aminotransferase, iron, and creatinine; DNA fragmentation and cell cycle of bone marrow cells; and liver, kidney and lung histology. In addition, the antitumor activity of rhodium (II) citrate and maghemite nanoparticles coated with rhodium (II) citrate was verified by tumor volume reduction, histology and immunohistochemistry.ResultsRegarding the treatments’ toxicity, no experimental groups had alterations in levels of serum ALT or creatinine, and this suggestion was corroborated by the histopathologic examination of liver and kidney of mice. Moreover, DNA fragmentation frequency of bone marrow cells was lower than 15% in all experimental groups. On the other hand, the complexes rhodium (II) citrate-functionalized maghemite and free rhodium (II) citrate led to a marked growth inhibition of tumor and decrease in CD31 and Ki-67 staining.ConclusionsIn summary, we demonstrated that both rhodium (II) citrate and maghemite nanoparticles coated with rhodium (II) citrate formulations exhibited antitumor effects against 4T1 metastatic breast cancer cell line following intratumoral administration. This antitumor effect was followed by inhibition of both cell proliferation and microvascularization and by tumor tissue injury characterized as necrosis and fibrosis. Remarkably, this is the first published report demonstrating the therapeutic efficacy of maghemite nanoparticles coated with rhodium (II) citrate. This treatment prolonged the survival period of treated mice without inducing apparent systemic toxicity, which strengthens its use for future breast cancer therapeutic applications.

Simultaneous inhibition of metastasis and growth of breast cancer by co-delivery of twist shRNA and paclitaxel using pluronic P85-PEI/TPGS complex nanoparticles

The metastasis of breast cancer is the leading cause of cancer death in women, and the lung is a common location of a secondary tumor that has metastasized from the primary source tumor. In this work, an attempt to simultaneously inhibit the metastasis and growth of tumor by co-delivering Twist shRNA (shTwi) and paclitaxel (PTX) using the conjugate of pluronic P85 (P85) and low molecular weight polyethyleneimine (PEI) (P85-PEI)/D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) complex nanoparticles (PTPNs) was performed on metastatic 4T1 breast cancer cell line and its pulmonary metastasis mice model. The experimental results demonstrated that PTPNs could effectively achieve cellular uptake and RNA interference. The down-regulation of Twist protein resulted in significant inhibitory effect of cell migration and invasion with the inhibition rate of 88.7% and 91.06%, respectively. The IC50 of PTPNs against 4T1 cells was 63-fold lower than that of free PTX. The prolonged circulation and increased accumulation of PTX and shTwi in lung and tumor were observed in in vivo biodistribution. The in vivo antitumor efficacy showed that PTPNs could not only inhibit the in situ tumor growth effectively, but also completely restrict the pulmonary metastasis in 4T1 pulmonary metastatic mice model. Therefore, co-delivering chemotherapy drugs with metastasis regulator by PTPNs to simultaneously inhibit metastasis and growth of tumor could achieve synergistic effect for the effective therapy of metastatic breast cancer.

Abstract 295: IDO drives tumor-promoting, pathogenic inflammation in lung

Abstract First identified as a mediator of acquired immune tolerance of the ‘foreign’ fetus from maternal immunity, the tryptophan-catabolizing enzyme IDO (indoleamine 2,3-dioxygenase) has since been implicated in tumor escape from the host immune system. Insight into the intricate role of IDO in the classical DMBA/TPA skin carcinogenesis model suggested that inflammatory tumor environments can induce IDO production resulting in de novo tumor development. In the genetically deficient model of IDO, mice show resistance to tumor formation. This provided a basis for our current studies exploring the importance of IDO in the microenvironment of the lung. To this end, we have investigated both primary tumor formation and metastatic disease in the lungs of IDO-deficient mice using the KRAS-induced lung adenocarcinoma and the metastatic 4T1 breast cancer models. Elevation of the inflammatory cytokine IL6 was associated with tumor outgrowth in the lungs in both models but was greatly attenuated with the loss of IDO, consistent with the in vitro demonstration that IDO activity markedly potentiates IL6 production. MDSCs (myeloid derived suppressor cells) exhibited reduced T cell suppressive activity when isolated from tumor-bearing, IDO-deficient animals that could be rescued by ectopic production of IL6 in the tumor. IL6 production could likewise reverse the pulmonary metastasis resistance exhibited by IDO-deficient mice. Interestingly, while there is a clear role of the immune system in lung tumor and metastatic outgrowth, IDO-deficient mice appear to have reduced vascularization in the lung which may partly contribute to reduced tumor formation. Together, these findings genetically validate IDO as a therapeutic target in the settings of pulmonary cancer and metastasis and establish the importance of IDO as a driver of IL6 production and MDSC function. Furthermore, the correlation of IDO to angiogenesis may be a new insight into the role of this enzyme in cancer. 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 295. doi:1538-7445.AM2012-295

Effects of magnetic induction hyperthermia and radiotherapy alone or combined on a murine 4T1 metastatic breast cancer model

Purpose: The purpose of this study was to explore the effects of MIH and radiotherapy alone or combined on metastatic breast cancer and the underlying mechanisms.Materials and methods: A murine 4T1 metastatic breast cancer model was established and randomly assigned into four treatment groups: C (control), R (radiotherapy), MIH, and MIH + R. Tumour volume, lung metastasis, the expression of Bax and MMP-9, T cell subsets, serum cytokine levels, and mouse survival were evaluated.Results: Group MIH + R showed significantly reduced tumour volume, lung metastasis, improved survival and increased Bax expression compared to group R or MIH (P < 0.05). MMP-9 expression in the primary tumour tissue was significantly increased in group R compared to the other groups (P < 0.05), which could be brought down by combined MIH treatment. Group MIH + R showed significantly higher CD4+ T cell percentage as well as CD4+/CD8+ cell ratio than group R (P < 0.05). Group MIH + R showed significantly higher serum levels of TNF-α, IFN-γ and IL-2 than group R (P < 0.05).Conclusions: MIH not only promotes the tumour-cell killing effect of radiotherapy through Bax-mediated cell death, but also improves cellular immunity in mice under radiotherapy and decreases the potential of radiotherapy to enhance MMP-9 expression, which leads to significant improvement in lung metastasis and overall survival of mice under combined treatment of MIH and R. This study is the first to have explored the effect of combined hyperthermia and radiotherapy on tumour metastasis and the underlying mechanisms. It provides insights into the application of MIH as an adjuvant to radiotherapy for metastatic breast cancer.

Increased Expression of P-Glycoprotein Is Associated with Doxorubicin Chemoresistance in the Metastatic 4T1 Breast Cancer Model

Development of drug resistance is one of the major causes of breast cancer treatment failure. The goal of this study was to understand the chemoresistance mechanism using the highly metastatic 4T1 breast cancer model, which emulates stage IV breast cancer in humans. The metastatic 4T1 breast cancer cell line treated with either doxorubicin or 5-FU showed a concentration-dependent reduced cell proliferation, with induced G2-phase growth arrest (doxorubicin) or G1-phase growth arrest (5-FU). Doxorubicin treatment partially suppressed the multiorgan metastasis of 4T1 breast cancer cells in the lung, heart, liver, and bone, compared with either 5-FU or cyclophosphamide. We isolated and characterized 4T1 breast cancer cells from doxorubicin-resistant metastatic tumors (cell line 4T1-R). Multiorgan metastasis of drug-resistant 4T1 breast tumors was totally resistant to doxorubicin treatment. Our results indicate that doxorubicin is localized exclusively in the cytoplasm of resistant 4T1 breast cancer cells and that it cannot reach the nucleus because of increased nuclear expression of P-glycoprotein. Pretreatment of doxorubicin-resistant 4T1-R breast cancer cells with verapamil, a general inhibitor of P-glycoprotein, increased nuclear translocation of doxorubicin and cellular cytotoxicity. Thus, impaired nuclear translocation of doxorubicin due to increased expression of P-glycoprotein is associated with doxorubicin resistance of highly metastatic 4T1 breast cancer.

Transforming growth factor-β-induced epithelial–mesenchymal transition facilitates epidermal growth factor-dependent breast cancer progression

TGF-β and EGF play critical roles in regulating the metastasis of aggressive breast cancers, yet the impact of epithelial-mesenchymal transition (EMT) induced by TGF-β in altering the response of breast cancer cells to EGF remains unknown. We show here that murine metastatic 4T1 breast cancer cells formed compact, and dense spheroids when cultured under 3-dimensional (3D) conditions, which contrasted sharply to the branching phenotypes exhibited by their nonmetastatic counterparts. Using the human MCF10A series, we show that epithelial-type and nonmetastatic breast cancer cells were unable to invade to EGF, while their mesenchymal-type and metastatic counterparts readily invaded to EGF. Furthermore, EMT induced by TGF-β was sufficient to manifest spheroid morphologies, a phenotype that increased primary tumor exit and invasion to EGF. Post-EMT invasion to EGF was dependent upon increased activation of EGFR and p38 MAPK, all of which could be abrogated either by pharmacological (PF-271) or genetic (shRNA) targeting of focal adhesion kinase (FAK). Mechanistically, EMT induced by TGF-β increased cell surface levels of EGFR and prevented its physical interaction with E-cadherin, leading instead to the formation of oncogenic signaling complexes with TβR-II. Elevated EGFR expression was sufficient to transform normal mammary epithelial cells, and to progress their 3D morphology from that of hollow acini to branched structures characteristic of nonmetastatic breast cancer cells. Importantly, we show that TGF-β stimulation of EMT enabled this EGFR-driven breast cancer model to abandon their inherent branching architecture and form large, undifferentiated masses that were hyper-invasive to EGF and displayed increased pulmonary tumor growth upon tail vein injection. Finally, chemotherapeutic targeting of FAK was sufficient to revert the aggressive behaviors of these structures. Collectively, this investigation has identified a novel EMT-based approach to neutralize the oncogenic activities of EGF and TGF-β in aggressive and invasive forms of breast cancer.

KLF17 is a negative regulator of epithelial–mesenchymal transition and metastasis in breast cancer

Metastasis is a complex multistep process, which requires the concerted action of many genes and is the primary cause of cancer death. Both pathways that regulate metastasis enhancement and those that regulate its suppression contribute to the tumour dissemination process. To identify new metastasis suppressors, we set up a forward genetic screen in a mouse model. We transduced a genome-wide RNA interference (RNAi) library into the non-metastatic 168FARN breast cancer cell line and orthotopically transplanted the cells into mouse mammary fat pads. We then selected cells that could metastasize to the lung and identified an RNAi for the KLF17 gene. Conversely, we demonstrate that ectopic expression of KLF17 in a highly metastatic 4T1 breast cancer cell line inhibits the ability of cells to metastasize from the mammary fat pad to the lung. We also show that suppression of KLF17 expression promotes breast cancer cell invasion and epithelial-mesenchymal transition (EMT), and that KLF17 protein functions by directly binding to the promoter region of Id1 (which encodes a key metastasis regulator in breast cancer) to inhibit its transcription. Finally, we demonstrate that KLF17 expression is significantly downregulated in primary human breast cancer samples and that the combined expression pattern of KLF17 and Id1 can serve as a potential biomarker for lymph node metastasis in breast cancer.

Preclinical Efficacy of Cystatin C to Target the Oncogenic Activity of Transforming Growth Factor β in Breast Cancer

We previously identified cystatin C (CystC) as a novel antagonist of transforming growth factor beta (TGF-beta) signaling in normal and malignant cells. However, whether the anti-TGF-beta activities of CystC can be translated to preclinical animal models of breast cancer growth and metastasis remains unproven. Assessing the preclinical efficacy of CystC was accomplished using metastatic 4T1 breast cancer cells, whose oncogenic responses to TGF-beta were inhibited both in vitro and in vivo. Indeed, we observed CystC to prevent TGF-beta from stimulating the growth and pulmonary metastasis of 4T1 tumors in mice in part by reducing the extent of Smad2, p38 mitogen-activated protein kinase, and extracellular signal-regulated kinase 1/2 phosphorylation present in 4T1 tumors. We also found CystC to significantly antagonize angiogenesis in developing 4T1 tumors, suggesting a novel role for CystC in uncoupling TGF-beta signaling in endothelial cells (ECs). Accordingly, CystC dramatically reduced murine and human EC responsiveness to TGF-beta, including their ability to regulate the expression of 1) TGF-beta signaling components, 2) inhibitor of differentiation (ID) family members, and 3) matrix metalloproteinases and their inhibitors (TIMPs) and to undergo cell invasion and angiogenic sprouting stimulated by TGF-beta. Importantly, CystC prevented TGF-beta from stimulating vessel development in Matrigel plugs implanted into genetically normal mice. Collectively, our findings provide the first preclinical evidence that CystC is efficacious in preventing breast cancer progression and angiogenesis stimulated by the oncogenic TGF-beta signaling system and suggest that CystC-based chemotherapeutics possesses translational efficacy to one day treat and improve the clinical course of late-stage breast cancers.

α-Tocopheryl Succinate as a Scaffold to Develop Potent Inhibitors of Breast Cancer Cell Adhesion

This study is aimed at the pharmacological exploitation of alpha-tocopheryl succinate (1) to develop potent antiadhesion agents. Considering the structural cooperativity between the phytyl chain and the carboxylic terminus in determining the antiadhesion activity, our structural optimization led to compound 5 ([2-(4,8-dimethyl-non-1-enyl)-2,5,7,8-tetramethyl-chroman-6-yloxy]-acetic acid), which exhibited an-order-of-magnitude higher potency than 1 in blocking the adhesion of 4T1 metastatic breast cancer cells to extracellular matrix proteins (IC(50), 0.6 microM versus 10 microM). Evidence indicates that the ability of compound 5 to block cell adhesion and migration was attributable to its effect on disrupting focal adhesion and actin cytoskeletal integrity by facilitating the degradation of focal adhesion kinase. Interactions between tumor cells and the ECM in the tumor microenvironment have been increasingly recognized as critical modulators of the metastatic potential of tumor cells. Consequently, the ability of compound 5 to block such interactions provides a unique pharmacological tool to shed light onto mechanisms that govern cell adhesion and tumor metastasis.

X-linked Inhibitor of Apoptosis Protein and Its E3 Ligase Activity Promote Transforming Growth Factor-Β-mediated Nuclear Factor-κB Activation during Breast Cancer Progression

The precise sequence of events that enable mammary tumorigenesis to convert transforming growth factor-beta (TGF-beta) from a tumor suppressor to a tumor promoter remains incompletely understood. We show here that X-linked inhibitor of apoptosis protein (xIAP) is essential for the ability of TGF-beta to stimulate nuclear factor-kappaB (NF-kappaB) in metastatic 4T1 breast cancer cells. Indeed whereas TGF-beta suppressed NF-kappaB activity in normal mammary epithelial cells, those engineered to overexpress xIAP demonstrated activation of NF-kappaB when stimulated with TGF-beta. Additionally up-regulated xIAP expression also potentiated the basal and TGF-beta-stimulated transcriptional activities of Smad2/3 and NF-kappaB. Mechanistically xIAP (i) interacted physically with the TGF-beta type I receptor, (ii) mediated the ubiquitination of TGF-beta-activated kinase 1 (TAK1), and (iii) facilitated the formation of complexes between TAK1-binding protein 1 (TAB1) and IkappaB kinase beta that enabled TGF-beta to activate p65/RelA and to induce the expression of prometastatic (i.e. cyclooxygenase-2 and plasminogen activator inhibitor-1) and prosurvival (i.e. survivin) genes. We further observed that inhibiting the E3 ubiquitin ligase function of xIAP or expressing a mutant ubiquitin protein (i.e. K63R-ubiquitin) was capable of blocking xIAP- and TGF-beta-mediated activation of NF-kappaB. Functionally xIAP deficiency dramatically reduced the coupling of TGF-beta to Smad2/3 in NMuMG cells as well as inhibited their expression of mesenchymal markers in response to TGF-beta. More importantly, xIAP deficiency also abrogated the formation of TAB1.IkappaB kinase beta complexes in 4T1 breast cancer cells, thereby diminishing their activation of NF-kappaB, their expression of prosurvival/metastatic genes, their invasion through synthetic basement membranes, and their growth in soft agar. Collectively our findings have defined a novel role for xIAP in mediating oncogenic signaling by TGF-beta in breast cancer cells.

Rejection of Metastatic 4T1 Breast Cancer by Attenuation of Treg Cells in Combination With Immune Stimulation

4T1 breast carcinoma is a highly malignant and poorly immunogenic murine tumor model that resembles advanced breast cancer in humans, and is refractory to most immune stimulation-based treatments. We hypothesize that the ineffectiveness of immune stimulatory treatment is mediated by the suppressive effects of CD4(+)CD25(+) regulatory T (Treg) cells, which can be attenuated by engaging the glucocorticoid-induced tumor necrosis factor receptor family-related protein with its natural ligand (GITRL); further, combination treatment with existing immune stimulation regimens will augment anti-tumor immunity and eradicate metastatic 4T1 tumors in mice.A soluble homodimeric form of mouse GITRL (mIg-mGITRLs) was molecularly constructed and used to treat orthotopic 4T1 tumors established in immune-competent, syngeneic Balb/c mice. When applied in combination with adenovirus-mediated intratumoral murine granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-12 (IL-12) gene delivery plus systemic 4-1BB activation, mIg-mGITRLs attenuated the immune-suppressive function of splenic Treg cells, which led to elevated interferon-gamma (IFN-gamma) production, tumor-specific cytolytic T-cell activities, tumor rejection and long-term survival in 65% of the animals without apparent toxicities. The results demonstrate that addition of mIg-mGITRLs to an immune-stimulatory treatment regimen significantly improved long-term survival without apparent toxicity, and could potentially be clinically translated into an effective and safe treatment modality for metastatic breast cancer in patients.

The Selectivity of Ethanolic Extract of Buah Makassar (Brucea javanica) on Metastatic Breast Cancer Cells

Treatment of cancer such as surgery, radiotherapy and chemotherapy has many side effects. Chemopreventive agent is needed to reduce the side effect and increase the effectivity of therapy. The discovery of cochemopreventive agent should consider on its selectivity to reduce side effects. The selective cochemopreventive agents work effectively in cancer cells and safe for normal cells. Buah Makassar (Brucea javanica) is a natural product that is empirically used for anti-inflammatory and antitumor. The purpose of this study is to determine the cytotoxic effect of ethanolic extract of buah Makassar against 4T1, MCF7, HeLa, and Vero cell lines. The cytotoxic test is performed by MTT assay. The parameter obtained from the cytotoxic test was IC50. Selectivity index is determined from IC50 ratio of cancer cells to normal cells. The results showed that ethanolic extract of buah Makassar has a cytotoxic activity on 4T1, MCF7, HeLa, and Vero cells with IC50 were 49,9±0,83 μg/mL; 107,6±8,14 μg/mL; 228,9±4,16 μg/mL and 395,5± 4,21 μg/mL respectively. It also has high selectivity on 4T1 metastatic breast cancer cell with selectivity index of 7,93. It can be concluded that the ethanolic extract of buah Makassar has potential to be delevoped as cochemopreventive agent especially on metastatic breast cancer. Keywords: Brucea javanica, MTT assay, selectivity index, 4T1, MCF7, HeLa, Vero