Metastatic Breast Carcinoma Cells Research Articles

Lobular Carcinoma of the Breast Metastatic to the Spleen and Accessory Spleen: Report of a Case.

Despite the fact that accessory spleen (also known as supernumerary spleen, splenunculus, or splenule) can be found in 10–30% of patients undergoing autopsies, metastatic disease occurring in this organ has been barely reported. A case of lobular breast carcinoma metastatic to the spleen and accessory spleen found incidentally at therapeutic splenectomy for severe anemia and thrombocytopenia is described. On microscopic examination both organs revealed severe fibrocongestive changes and extramedullary hematopoiesis with no obvious carcinomatous involvement. Cytokeratin 7, estrogen receptors, and GATA3 immunohistochemistry disclosed the presence of numerous metastatic breast carcinoma cells infiltrating the splenic parenchyma. This case demonstrates that metastatic carcinoma can be encountered, although rarely, in accessory spleens and that cytokeratin stain should be performed in sections of spleens and/or accessory spleens excised from cancer patients in which the presence of malignant epithelial cells is not recognized on routine sections.

Abstract 332: Extracellular matrix stiffness regulates metabolic state in metastatic, but not quiescent, breast carcinoma cells

Abstract Increased breast density is associated with a 4-6 fold increased risk of developing breast cancer, and is associated with an increase in deposition of extracellular matrix (ECM) proteins, most abundantly collagen I. Collagen, like other ECM proteins, plays a structural role fundamental to tissue organization. High levels of collagen deposition correspond with a stiffer ECM, which is emerging as an important regulator of cell proliferation and tumor progression. Our previously reported microarray implicated changes in mammary epithelial cell metabolism in response to increased matrix stiffness, consistent with the expanding role of the ECM in tumor cell signaling. Here we report that increased matrix stiffness regulates the expression of pyruvate dehydrogenase kinase 1, a key regulator between lactic acid production and pyruvate entry into the mitochondria, in highly metastatic 4T1 breast carcinoma cells. Interestingly, we do not observe this same metabolic regulation in quiescent (dormant) 4T07 tumor cells of the same lineage. These alterations in protein expression correlated to changes in cellular NADH metabolism observed by several independent approaches, including metabolic flux analysis and quantitative imaging fluorescence lifetime microscopy. Thus, we find that alterations in collagen stiffness cause metabolic shifts between oxidative phosphorylation and aerobic glycolysis in highly metastatic cells, but not in quiescent cells. These findings identify stiffness of the ECM as an important regulator of metabolic state, and further identify quiescence as a dominant trait that is not overcome by ECM stiffness. Citation Format: Brett A. Morris, Brian Burkel, Suzanne Ponik, Kevin Eliceiri, Julio Aguirre-Ghiso, John Condeelis, James Castracane, Patricia J. Keely. Extracellular matrix stiffness regulates metabolic state in metastatic, but not quiescent, breast carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 332. doi:10.1158/1538-7445.AM2015-332

HGF and TGFβ1 differently influenced Wwox regulatory function on Twist program for mesenchymal-epithelial transition in bone metastatic versus parental breast carcinoma cells.

BackgroundMuch effort has been devoted to determining how metastatic cells and microenvironment reciprocally interact. However, the role of biological stimuli of microenvironment in controlling molecular events in bone metastasis from breast carcinoma for mesenchymal-epithelial transition (MET) is largely unknown. The purpose of the present paper was to clarify (1) the influence of hepatocyte-growth factor (HGF) and transforming growth factorβ1 (TGFβ1) on the phenotype of bone-metastatic 1833 and parental MDA-MB231 cells; (2) the hierarchic response of Twist and Snail controlled by Wwox co-factor, that might be critical for the control of 1833-adhesive properties via E-cadherin.MethodsWe studied under HGF and TGFβ1 the gene profiles—responsible for epithelial-mesenchymal transition (EMT), versus the revertant MET phenotype—making the correspondence with 1833 morphology and the relation to HGF-dependent control of TGFβ1 signalling. In particular, the activation of Twist program and the underlying molecular mechanisms were investigated, considering the role of endogenous and exogenous Wwox with siRNAWWOX and the expression vector transfection, to clarify whether Twist affected E-cadherin transactivation through a network of transcription factors and regulators.ResultsHGF and TGFβ1 oppositely affected the expression of Wwox in 1833 cells. Under HGF, endogenous Wwox decreased concomitant with Twist access to nuclei and its phosphorylation via PI3K/Akt pathway. Twist activated by HGF did not influence the gene profile through an E-box mechanism, but participated in the interplay of PPARγ/Ets1/NF-kB-transcription factors, triggering E-cadherin transactivation. Altogether, HGF conferred MET phenotype to 1833 cells, even if this was transient since followed by TGFβ1-signalling activation. TGFβ1 induced Snail in both the cell lines, with E-cadherin down-regulation only in 1833 cells because in MDA-MB231 cells E-cadherin was practically absent. Exogenous Wwox activated metastatic HIF-1, with Twist as co-factor.ConclusionsHGF and TGFβ1 of bone-metastasis microenvironment acted co-ordinately, influencing non redundant pathways regulated by Twist program or Snail-transcription factor, with reversible MET switch. This process implicated different roles for Wwox in the various steps of the metastatic process including colonization, with microenvironmental/exogenous Wwox that activated HIF-1, important for E-cadherin expression. Interfering with the Twist program by targeting the pre-metastatic niche stimuli could be an effective anti-bone metastasis therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0389-y) contains supplementary material, which is available to authorized users.

Enhanced antitumor effects of the BRBP1 compound peptide BRBP1-TAT-KLA on human brain metastatic breast cancer.

Novel molecularly targeted agents that block the development and metastasis of human brain metastatic breast cancer hold great promise for their translational value. In this study, we constructed a novel targeting composite peptide BRBP1-TAT-KLA comprising of three elements: a brain metastatic breast carcinoma cell (231-BR)-binding peptide BRBP1, a cell penetrating peptide TAT, and a proapoptotic peptide KLA. This composite peptide efficiently internalized in 231-BR cells and consequently induced mitochondrial damage and cellular apoptosis. Exposure of 231-BR cells to BRBP1-TAT-KLA significantly decreased cell viability and increased apoptosis compared with the cells treated with the control peptides. In vivo relevance of these findings was further corroborated in the 231-BR tumor-bearing mice that demonstrated significantly delayed tumor development and metastasis following administration of BRBP1-TAT-KLA compared with those treated with TAT-KLA alone. Interestingly, BRBP1-TAT-KLA inhibited the formation of both large and micro-metastases, while TAT-KLA alone failed to significantly reduce micro-metastases in the breast cancer brain metastasis mice. BRBP1-TAT-KLA selectively homed to the tumors in vivo where it induced cellular apoptosis without significant toxicity on non-tumor tissues. Our findings therefore demonstrated the enhanced antitumor effects of the BRBP1 compound peptide BRBP1-TAT-KLA, providing insights toward development of a potential therapeutic strategy for brain metastatic breast cancer.

Abstract 1131: Snail- and ERK2-dependent signaling enhances breast cancer cell resistance to hydroxytamoxifen

Abstract Snail transcription factor and MAPK/ERK signaling regulate EMT and chemotherapy resistance in various tumor models by binding to target promoters (i.e., E-cadherin, maspin, ER-α). ERK1 is expressed during embryogenesis and in non-metastatic cells; ERK2 is implicated during vasculogenesis and promotes stem cell phenotype in triple negative breast cancer. Nuclear-localized ERK is associated with more active and potentially metastatic breast and ovarian carcinoma cells; cytoplasmic-localized ERK is a good prognostic factor. The role that Snail plays during the transition from cytoplasmic ERK1 to nuclear ERK2 has not been investigated. We hypothesized that both nuclear Snail and ERK2 are required for breast cancer cells to become resistant to 4-hydroxytamoxifen (4-OHT). We utilized MCF-7 breast adenocarcinoma cells, MCF-7 transfected stably with empty Neo vector control (MCF-7 Neo) or constitutively active Snail cDNA (MCF-7 Snail). MCF-7 Neo and MCF-7 Snail have previously represented a breast cancer EMT model; we used this model to analyze ERK signaling and therapy response. Preliminary data suggest that MCF-7 Neo express higher levels of p-ERK, ER-α, and E-cadherin and no Snail. MCF-7 Snail expressed higher levels of Snail and vimentin, low levels of p-ERK, and low ER-α and no E-cadherin. MCF-7 Neo expressed higher ERK1 and MCF-7 Snail expressed higher ERK2. Immunocytochemistry indicated that p-ERK was nuclear and cytoplasmic in MCF-7 Neo, but mostly nuclear in MCF-7 Snail. We treated MCF-7 Neo and MCF-7 Snail with control siRNA, ERK1 siRNA, or ERK2 siRNA, then determined the expression of Snail, p-ERK1/2, ERK1/2, E-cadherin, and ER-α. We found that ERK1 siRNA decreased E-cadherin, while ERK2 siRNA increased E-cadherin in MCF-7 Neo. ERK2 siRNA decreased Snail, but ERK1 siRNA had no effect on Snail. ERK1 siRNA increased active ERK2 and ERK2 siRNA increased ERK1 in MCF-7 Neo and MCF-7 Snail. To determine the role that Snail plays in resistance, we treated MCF-7 Neo and MCF-7 Snail with 4-OHT and/or UO126 then mitochondrial permeability and caspase-3/7 activity assays. MCF-7 Neo had higher mitochondrial permeability than MCF-7 Snail, indicative of the dimmer staining using TMRM stain, and MCF-7 Neo had higher caspase-3/7 activity after treatments. Overall, cell death in MCF-7 Snail was caspase-independent. These data imply that Snail and ERK2 collaborate through an unknown mechanism to promote EMT and therapy resistance in breast cancer cells. Targeting Snail and/or ERK2 in advanced breast cancer may be a potential therapeutic or preventive strategy. Citation Format: Bethany N. Smith, Peri Nagappan, Latonia Taliaferro-Smith, Roman Mezencev, Clayton Yates, Cimona Hinton, Valerie Odero-Marah. Snail- and ERK2-dependent signaling enhances breast cancer cell resistance to hydroxytamoxifen. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1131. doi:10.1158/1538-7445.AM2014-1131

Intratumoral heterogeneity impacts the response to anti-neu antibody therapy.

BackgroundAlong with de novo resistance, continued exposure to trastuzumab, an anti-human epidermal growth factor receptor 2 (HER2/neu) antibody, can lead to acquired resistance. In this study, we characterize a new anti-HER2/neu antibody resistant and metastatic mouse breast carcinoma cell line, TUBO-P2J. This cell line was developed during in vivo experiments using the antibody sensitive and non-metastatic tumor line TUBO. In addition, TUBO-P2J was used to establish an intratumoral HER2 heterogenous animal tumor model to evaluate the therapeutic effects of anti-HER2/neu antibody.MethodsAfter establishing the cell line, TUBO-P2J was characterized regarding its susceptibility to anti-neu antibody and chemotherapeutics, as well as its metastatic potential in vitro and in vivo. In addition, expression profiles of metastasis related genes were also evaluated. A clinically relevant intratumoral HER2 heterogenous tumor model was established by inoculating mice with tumor cells consisting of TUBO and TUBO-P2J at a ratio of 1,000:1 or 10,000:1. Tumor growth and mouse survival were used to evaluate the therapeutic effects of anti-neu antibody.ResultsThe TUBO-P2J cell line is a HER2/neu negative and highly metastatic variant of TUBO. This cell line was resistant to anti-neu antibody therapy, and when inoculated subcutaneously, metastasized to the lungs within 14 days. Compared to the parental TUBO cell line, TUBO-P2J displayed an epithelial-mesenchymal transition (EMT) related gene expression profile including: the loss of E-cadherin, and increased Vimentin, Snail, and Twist1 expression. In addition, TUBO-P2J exhibited increased invasion and migration activity, and was resistant to chemotherapy drugs. Finally, mixed tumor implantations experiments revealed that an increased percentage of TUBO-P2J rendered tumors less responsive to anti-neu antibody therapy.ConclusionThis study describes a novel model of intratumoral heterogenous metastatic breast cancer in immune competent mice that can be used to develop novel or combined immunotherapies to overcome antibody resistance.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-647) contains supplementary material, which is available to authorized users.

Panax notoginseng saponins (PNS) inhibits breast cancer metastasis

Ethnopharmacological relevancePanax notoginseng (Burkill) F.H. Chen (Araliaceae) has been extensively used as a therapeutic agent to treat a variety of diseases. Panax notoginseng saponins (PNS) consist of major therapeutically active components of Panax notoginseng. PNS inhibit the growth of a variety of tumor cells in vitro and in vivo. The aim of the study is to investigate the effects and underlying mechanisms of PNS on breast cancer metastasis. Materials and methods4T1 cell, a highly metastatic mouse breast carcinoma cell line, was utilized for in vitro and in vivo assays. In vitro assays were first performed to examine the effects of PNS on 4T1 cell viability, migration and invasion, respectively. Real-time PCR analyses were also performed to examine the effects of PNS on the expression of genes associated with tumor metastasis. The effect of PNS on 4T1 tumor cell metastasis was further assessed in spontaneous and experimental metastasis models in vivo. ResultsPNS treatment exhibited a dose-dependent effect on impairing 4T1 cell viability in vitro. However, when examined at a lower dose that did not affect cell viability, the migration and invasion of 4T1 cell was remarkably inhibited in vitro. Meanwhile, PNS treatment led to upregulated expression of genes known to inhibit metastasis and downregulated expression of genes promoting metastasis in cultured 4T1 cells. These results suggested a selective effect of PNS on 4T1 migration and invasion. This hypothesis was further addressed in 4T1 metastasis models in vivo. The results showed that the lung metastasis was significantly inhibited by PNS treatment in both spontaneous and experimental metastasis models. ConclusionTaken together, our results demonstrated an inhibitory effect of PNS on 4T1 tumor metastasis, warranting further evaluation of PNS as a therapeutic agent for treating breast cancer metastasis.

Parthenolide and costunolide reduce microtentacles and tumor cell attachment by selectively targeting detyrosinated tubulin independent from NF-κB inhibition

IntroductionDetyrosinated tubulin, a post-translational modification of α-tubulin and a hallmark of stable microtubules, has gained recent attention given its association with tumor progression, invasiveness, and chemoresistance. We also recently reported that epithelial-to-mesenchymal transition (EMT) promotes tubulin detyrosination through tubulin tyrosine ligase (TTL) suppression. Furthermore, detyrosinated tubulin-enriched membrane protrusions, termed microtentacles (McTN), facilitate tumor cell reattachment to endothelial layers. Given the induction of EMT associated with inflammation and cancer progression, we tested anti-inflammatory nuclear factor-kappaB (NF-κB) inhibitors on a panel of human breast carcinoma cells to examine their effects on detyrosinated tubulin to identify more specific tubulin-directed anti-cancer treatments.MethodsUsing metastatic human breast carcinoma cells MDA-MB-157, MDA-MB-436, and Bt-549, we measured the impact of NF-κB inhibitors parthenolide, costunolide, and resveratrol on detyrosinated tubulin using protein expression analysis and immunofluorescence. A luciferase reporter assay and a viability screen were performed to determine if the effects were associated with their NF-κB inhibitory properties or were a result of apoptosis. Real-time monitoring of cell-substratum attachment was measured utilizing electrical impedance across microelectronic sensor arrays. We compared the selectivity of the NF-κB inhibitors to specifically target detyrosinated tubulin with traditional tubulin-targeted therapeutics, paclitaxel and colchicine, throughout the study.ResultsSesquiterpene lactones, parthenolide and costunolide, selectively decrease detyrosinated tubulin independent of their inhibition of NF-κB. Live-cell scoring of suspended cells treated with parthenolide and costunolide show reduction in the frequency of microtentacles and inhibition of reattachment. Structural analysis shows that parthenolide and costunolide can decrease detyrosinated microtubules without significantly disrupting the overall microtubule network or cell viability. Paclitaxel and colchicine display indiscriminate disruption of the microtubule network.ConclusionsOur data demonstrate that selective targeting of detyrosinated tubulin with parthenolide and costunolide can reduce McTN frequency and inhibit tumor cell reattachment. These actions are independent of their effects on NF-κB inhibition presenting a novel anti-cancer property and therapeutic opportunity to selectively target a stable subset of microtubules in circulating tumor cells to reduce metastatic potential with less toxicity in breast cancer patients.

Prolyl isomerase Pin1 negatively regulates the stability of SUV39H1 to promote tumorigenesis in breast cancer

Pin1, a conserved eukaryotic peptidyl-prolyl cis/trans isomerase, has profound effects on numerous key-signaling molecules, and its deregulation contributes to disease, particularly cancer. Although Pin1-mediated prolyl isomerization of protein servers as a regulatory switch in signaling pathways, the significance of proline isomerase activity in chromatin modifying complex remains unclear. Here, we identify Pin1 as a key negative regulator for suppressor of variegation 3-9 homologue 1 (SUV39H1) stability, a major methyltransferase responsible for histone H3 trimethylation on Lys9 (H3K9me3). Pin1 interacts with SUV39H1 in a phosphorylation-dependent manner and promotes ubiquitination-mediated degradation of SUV39H1. Consequently, Pin1 reduces SUV39H1 abundance and suppresses SUV39H1 ability to induce H3K9me3. In contrast, depletion of Pin1 in cancer cells leads to elevated SUV39H1 expression, which subsequently increases H3K9me3, inhibiting tumorigenecity of cancer cells. In a xenograft model with 4T1 metastatic mouse breast carcinoma cells, Pin1 overexpression increases tumor growth, whereas SUV39H1 overexpression abrogates it. In human breast cancer patients, immunohistochemical staining shows that Pin1 levels are negatively correlated with SUV39H1 as well as H3K9me3 levels. Thus, Pin1-mediated reduction of SUV39H1 stability contributes to convey oncogenic signals for aggressiveness of human breast cancer, suggesting that Pin1 may be a promising drug target for anticancer therapy.

Metastatic breast carcinoma induces vascular endothelial dysfunction in Balb-c mice: Role of the tumor necrosis factor-α and NADPH oxidase

Although the oxidative stress and inflammation are closely related with breast cancer, there is no study directly examining the possible changes in vascular functions in the presence of breast carcinoma. The goal of the present study was to evaluate changes in vascular reactivity in tumor-bearing mice. In this study, highly metastatic breast carcinoma cells which were derived from liver or brain metastasis of 4T1 murine breast carcinoma (4TLM and 4TBM, respectively), and 67NR cells which were tumorigenic but non-metastatic cells were used. Female Balb-c mice 8–10weeks old were divided into following groups: (1) control, (2) injected with 67NR, (3) injected with 4TLM, and (4) injected with 4TBM orthotopically. Thoracic aorta was removed 23–25days after injection of tumor cells. Isometric tension studies were performed in response to potassium chloride (KCl), phenylephrine (Phe), acetylcholine (ACh, an endothelium-dependent vasodilator), and sodium nitroprusside (SNP, an endothelium-independent vasodilator). Endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (Ser 1177) (p-eNOS), gp91phox, and tumor necrosis factor-α (TNF-α) expressions in aortic tissues were demonstrated by immunohistochemistry. The level of TNF-α in vascular tissue was measured by ELISA. The presence of tumor was resulted in significant inhibition of response to ACh in both 4TLM and 4TBM injected mice, but not 67NR injected mice. Furthermore, both KCl and Phe-induced contraction of thoracic aorta was not changed significantly in tumor-bearing animals. eNOS and p-eNOS expressions decreased while gp91phox and TNF-α expressions increased in endothelium of 4TLM and 4TBM mice compared to 67NR injected and control mice. Moreover, TNF-α levels of thoracic aorta in mice with metastatic breast carcinoma were significantly higher than that of 67NR mice. Tumor-induced endothelial dysfunction determined by ACh-induced relaxation improved by superoxide dismutase (SOD), apocynin (a NADPH oxidase inhibitor), and infliximab (a TNF-α monoclonal antibody). The findings of this study suggest that the presence of metastatic breast carcinoma may cause a significant reduction in endothelium-dependent relaxation of thoracic aorta via NADPH oxidase-mediated oxidative stress and TNF-α production.

Abstract 3288: Isolation of anti-neoplastic drug candidates against metastatic breast, lungs and ovarian carcinoma cells.

Abstract Cancer is a leading cause of death in man and women worl-wide. In recent years, wold-wide increase of obesity and diabetes, consumption of food additives and pollution are affecting quality of life and the incidense of all types of cancers are on the rise. Although new treatment regimens are also introduced into clinic war against cancer is still not won. Treatment of cancer with synthetic chemoterapics come with a unfavorable side effects which often ends therapy. Instead of investing in synthetic compounds, OZES BIOPHARMA is engaged in discovery of anti-neoplastic drug candidates from endemic plants of TURKEY. We have been extracting and analyzing organic compounds from variety of plants consumed as a food. Our search in past two years yielded very enchoraging results and we have discovered 6 novel compounds named OZS 1-6. Our compounds show significant levels of anti-tumor and anti-metastatic activities against human metastatic and non-metastatic breast cancer cells(MDA-231,MCF7), lung cancer cells (A549), Ovarian cancer cells (A2780) and its cisplatin resistant clone. Currently, we are idientifying the pathways inhibited or activated by our compounds. Citation Format: Osman N. Ozes. Isolation of anti-neoplastic drug candidates against metastatic breast, lungs and ovarian carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3288. doi:10.1158/1538-7445.AM2013-3288

BRMS1 Sensitizes Breast Cancer Cells to ATP-Induced Growth Suppression

Purinergic signaling may represent an effective target in cancer therapy because the expression of purinergic receptors is altered in many forms of cancer and extracellular nucleotides modulate cancer cell growth. We examined the effect of extracellular ATP on the growth of the metastatic breast carcinoma cell line MDA-MB-435 relative to an immortalized breast epithelial cell line, hTERT-HME1. We also investigated whether the metastasis suppressor gene BRMS1 alters the sensitivity of breast cancer cells to ATP. Exposure to ATP for 24 h decreased proliferation and induced apoptosis in hTERT-HME1. However, exposure to ATP did not decrease proliferation or induce apoptosis in MDA-MD-435 cells until 48 h of exposure and only at higher doses than were effective with hTERT-HME1, suggesting MDA-MB-435 cells were resistant to the antiproliferative and apoptosis-inducing effects of ATP. Exposure to ATP for 24 h induced a decrease in proliferation of MDA-MB-435 cells expressing BRMS1, similar to hTERT-HME1, but did not induce an increase in apoptosis. MDA-MB-435 cells expressed low levels of the purinergic receptor P2Y2, as well as decreased ATP-induced cytosolic calcium mobilization, relative to hTERT-HME1. However, expressing BRMS1 in MDA-MB-435 cells restored P2Y2 levels and ATP-induced cytosolic calcium mobilization such that they were similar to hTERT-HME1. These data suggest that BRMS1 increases the sensitivity of breast cancer cells to the antiproliferative, but not apoptosis-inducing effects of ATP and that this is at least partly mediated by increased expression of the P2Y2 receptor.

Nox4-dependent ROS modulation by amino endoperoxides to induce apoptosis in cancer cells

Tumor metastasis is the main cause of death in cancer patients. Anoikis resistance is one critical malefactor of metastatic cancer cells to resist current clinical chemotherapeutic treatments. Although endoperoxide-containing compounds have long been suggested as anticancer drugs, few have been clinically employed due to their instability, complex synthesis procedure or low tumor cell selectivity. Herein, we describe a one-pot strategy to synthesize novel amino endoperoxides and their derivatives with good yields and stabilities. In vitro cell-based assays revealed that 4 out of the 14 amino endoperoxides selectively induce metastatic breast carcinoma cells but not normal breast cells to undergo apoptosis, in a dose-dependent manner. Mechanistic studies showed that the most potent amino endoperoxide, 4-Me, is selective for cancer cells expressing a high level of Nox4. The anticancer effects are further shown to be associated with reduced O2−:H2O2 ratio and increased ·OH level in the cancerous cells. Animal study showed that 4-Me impairs orthotopic breast tumor growth as well as tumor cell metastasis to lymph nodes. Altogether, our study suggests that anticancer strategies that focus on redox-based apoptosis induction in tumors are clinically viable.

Abstract A11: Cancer cell-secreted CXCL1 chemokine acts on neutrophils to support metastasis

Abstract Innate immune cells such as macrophages, monocytes and neutrophils can be recruited to tumors by chemokines, which act on chemokines receptors on these cells. Macrophages and monocytes have well-established roles in promotion of metastasis. However, neutrophils have been reported to have both pro- and anti-metastatic effects. In this study, we used the metastatic 4T1 and non-metastatic 4T07 murine breast carcinoma cells lines (originating from the same tumor) to study effects of neutrophils in breast metastasis. Primary tumors from 4T1 cells, orthotopically injected into syngeneic mice, had more infiltrating neutrophils than did primary tumors from 4T07 cells. The increased numbers of neutrophils, which expressed the chemokine receptor CXCR2, was associated with significantly higher levels of cancer cell-secreted CXCL1, a chemokine ligand for CXCR2. To test whether cancer cell-secreted CXCL1 acted on neutrophils to regulate metastasis, we used shRNAs to reduce CXCL1 expression in the 4T1 cells. Consistent with the lack of CXCR2 expression by the cancer cells, this did not influence cancer cell proliferation or migration in vitro. However, in vivo, tumor infiltration of neutrophils and lung metastasis were both reduced. Moreover, when parental 4T1 cells were orthotopically injected into syngeneic Cxcr2-/-mice, neutrophil infiltration and lung metastasis were significantly decreased as compared to tumors in wild type littermate. These results suggest that paracrine CXCL1 by cancer cells facilitates neutrophil infiltration via CXCR2 and that neutrophils in turn promote lung metastasis. To examine the mechanisms by which interactions between cancer cells and neutrophils might promote lung metastasis, we performed co-culture experiments with cancer cells and neutrophils. We found that neutrophils directly stimulated 4T1 cell invasion through matrigel. Interestingly, cancer-released CXCL1 also promoted the formation of neutrophil extraceullar traps (NETs), which is an expulsion of DNA and proteases from the neutrophils. Both invasion and NET formation were blocked by inhibition of phagocyte NADPH oxidase. Moreover, when mice with 4T1 tumors were treated with a phagocyte NADPH oxidase inhibitor, tumor growth and lung metastasis was reduced. Finally, decreased levels of the phosphorylated form of p47phox, a cytosolic component of phagocyte NAPDH oxidase that is activated by phosphorylation, was decreased in the tumor lysates from 4T1 injected Cxcr2-/- mice as compared to Cxcr2+/- mice. In conclusion, our study revealed that cancer cell-secreted CXCL1 regulates neutrophil infiltration and extracellular trap formation, and that phagocyte NADPH oxidase activity in these neutrophils promoted cancer cell invasion and metastasis. Citation Format: Juwon Park, Jing Qiu, Elizabeth S. Nakasone, Mikala Egeblad. Cancer cell-secreted CXCL1 chemokine acts on neutrophils to support metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A11.

Immune stimulating photoactive hybrid nanoparticles for metastatic breast cancer

A therapeutic technology that combines the phototoxic and immune-stimulating ability of photodynamic therapy (PDT) with the widespread effectiveness of the immune system can be very promising to treat metastatic breast cancer. We speculated that the knowledge of molecular mechanisms of existing multi-component therapies could provide clues to aid the discovery of new combinations of an immunostimulant with a photosensitizer (PS) using a nanoparticle (NP) delivery platform. Therapeutic challenges when administering therapeutic combinations include the choice of dosages to reduce side effects, the definitive delivery of the correct drug ratio, and exposure to the targets of interest. These factors are very difficult to achieve when drugs are individually administered. By combining controlled release polymer-based NP drug delivery approaches, we were able to differentially deliver zinc phthalocyanine (ZnPc) based PS to metastatic breast cancer cells along with CpG-ODN, a single-stranded DNA that is a known immunostimulant to manage the distant tumors in a temporally regulated manner. We encapsulated ZnPc which is a long-wavelength absorbing PS within a polymeric NP core made up of poly(d,l-lactic-co-glycolic acid)-b-poly(ethylene glycol) (PLGA-b-PEG). After coating the outside of the polymeric core with gold NPs (AuNPs), we further modified the AuNP surface with CpG-ODN. In vitro cytotoxicity using 4T1 metastatic mouse breast carcinoma cells shows significant photocytotoxicity of the hybrid NPs containing both ZnPc and CpG-ODN after irradiation with a 660 nm LASER light and this activity was remarkably better than either treatment alone. Treatment of mouse bone marrow derived dendritic cells with the PDT-killed 4T1 cell lysate shows that the combination of PDT with a synergistic immunostimulant in a single NP system results in significant immune response, which can be used for the treatment of metastatic cancer.

Effects of radiotherapy and substance P on MIP-2 (macrophage inflammatory protein-2) levels in metastatic breast carcinoma cell lines.

e21130 Background: Breast cancer is ranked second among the most common cancers in the world. Radiotherapy (RT) is known to reduce the risk of the recurrence in cancer treatment. Substance P (SP), binds to the Neurokinin-1 (NK1) receptor which plays a role in inflammation, proliferation, angiogenesis and migration in cancer cells. MIP-2 (Macrophage inflammatory protein 2, IL-8 in human which has been currently understood to play a role in carcinogenesis is an inflammatory CXC chemokine. The aim of this study is to show whether MIP-2 levels alter with RT and whether SP has a role in this alteration. Methods: In this study, we investigated the effects of SP, NK1 receptor antagonist (NK1 RA) and RT on cell proliferation and MIP-2 secretion in 4T1 cells. 4T1 cells were derived from spontaneously formed breast tumors in Balb-c mice. We have cultured its subsets from metastatic lesions, and we named them as 4THM (heart metastasis), 4TLM (liver metastasis), 4TBM (brain metastasis). Cells were plated as 400-500 cells per well in a 96 well plate containing medium with 5% FBS. After 24-48 hours, cells were applied NK1RA and SP. After 3-4 hours of treatment, the group of RT was irradiated with 45 Gy. The proliferation of the cells was evaluated 48 hours after treatment. This proliferation was evaluated using WST cell proliferation reagent (WST-1, Roche). MIP-2 levels were measured with ELISA. Results: We observed that RT reduced the proliferation of cells in a ratio of 30,2 % in 4T1 cells, 43,9 % in 4THM cells, 44,7 % in 4TLM cells and 34 % in 4TBM cells. 4THM, 4TBM cells have the highest MIP-2 secretion, which was further increased after RT. Also, it was observed that SP, 10-8 M concentration decreased the RT-induced MIP-2 secretion. Conclusions: Increasing MIP-2 secretiondue to RT can precipitate cancer development and SP can be used as a therapeutic aim to prevent such an increase.

Abstract 3308: Axl is required for EMT-induced stem cell traits and metastasis in breast cancer

Abstract Epithelial-to-mesenchymal transition (EMT) endows carcinoma cells with migratory, survival and stem cell-like attributes that facilitate metastasis. We have shown that expression of the receptor tyrosine kinase Axl in primary breast cancer is a strong independent predictor of poor overall survival and is further enhanced in matched patient metastases. Axl is upregulated by EMT-induction in mammary epithelial cells and establishes an autocrine signaling loop with its ligand, Gas6. Axl signaling is required to maintain the mesenchymal and stem cell-like phenotype induced by EMT-transcription factors. Axl-inhibition downregulated mesenchymal and stem cell marker expression, blocked invasiveness, inhibited mammosphere formation and reversed drug resistance. Axl knockdown completely prevented the spread of metastatic breast carcinoma cells from the mammary gland and increased overall survival in two different orthotopic breast cancer models. Axl was also required for breast cancer cells to extravasate into the lung following intravenous injection. These results suggest that Axl signaling is necessary for metastasis, stem cell and therapy resistance traits in breast cancer. Hence, Axl-inhibition represents a novel therapeutic opportunity to target stem-like and drug resistant breast cancer cells. 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 3308. doi:1538-7445.AM2012-3308

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Determining β2-Integrin and Intercellular Adhesion Molecule 1 Binding Kinetics in Tumor Cell Adhesion to Leukocytes and Endothelial Cells by a Gas-driven Micropipette Assay

Interactions between polymorphonuclear neutrophils (PMNs) and tumor cells have been reported to facilitate the adhesion and subsequent extravasation of tumor cells through the endothelium under blood flow, both of which are mediated by binding β(2)-integrin to intercellular adhesion molecule 1 (ICAM-1). Here the adhesions between human WM9 metastatic melanoma cells, PMNs, and human pulmonary microvascular endothelial cells (HPMECs) were quantified by a gas-driven micropipette aspiration technique (GDMAT). Our data indicated that the cellular binding affinity of PMN-WM9 pair was 3.9-fold higher than that of the PMN-HPMEC pair. However, the effective binding affinities per molecular pair were comparable between the two cell pairs no matter whether WM9 cells or HPMECs were quiescent or cytokine-activated, indicating that the stronger adhesion between PMN-WM9 pair is mainly attributed to the high expression of ICAM-1 on WM9 cells. These results proposed an alternative mechanism, where WM9 melanoma cells adhere first with PMNs near vessel-wall regions and then bind to endothelial cells via PMNs under blood flow. In contrast, the adhesions between human MDA-MB-231 metastatic breast carcinoma cells and PMNs showed a comparable cellular binding affinity to PMN-HPMEC pair because the ICAM-1 expressions on MDA-MB-231 cells and HPMECs are similar. Furthermore, differences were observed in the intrinsic forward and reverse rates of the β(2)-integrin-ICAM-1 bond between PMN-TC and PMN-EC pairs. This GDMAT assay enables us to quantify the binding kinetics of cell adhesion molecules physiologically expressed on nucleated cells. The findings also further the understanding of leukocyte-facilitated tumor cell adhesion from the viewpoint of molecular binding kinetics.

Leptin upregulates tissue factor expression in human breast cancer MCF-7 cells

IntroductionObesity is a risk factor for both cardiovascular disease and cancer development. Leptin, a cytokine produced by adipose tissue, controls different processes in peripheral tissues, including cancer development and thrombotic disorders in patients with a variety of clinical disorders. Tissue factor (TF), the trigger of blood clotting, is abundant in the adipose tissue.Since TF, often expressed by cancer cells, is considered a hallmark of cancer progression, we investigated whether leptin could modulate TF in the human metastatic breast carcinoma cell line MCF-7. Materials and MethodsMCF-7 cells were incubated with or without the different reagents at 37°C. At the end of incubation, cells were tested for procoagulant activity by a one-stage clotting assay, TF and TNF-α antigen levels and mRNA by ELISA and real-time RT-PCR, respectively. Leptin receptor was studied by FACS. ResultsBoth TF activity and antigen constitutively expressed by MCF-7 were significantly increased by leptin in a dose-dependent fashion. TF mRNA levels were also enhanced indicating that leptin exerts its effect at the transcription level. The effect of leptin was specific and required binding to its receptor (Ob-R), which was found on the surface of the cells, since antibodies against leptin and Ob-R completely prevented TF expression upregulation.In addition, leptin enhanced both TNF-α mRNA synthesis and secretion from MCF7. An anti-TNF-α MoAb completely abolished the leptin-induced TF expression. ConclusionsThese data support the hypothesis that leptin, by its upregulation of TF, possibly mediated by TNF-α synthesis, may contribute to processes underlying both cancer and vascular cell disorders.