Lung Epithelial Cells Induce Both Phenotype Alteration and Senescence in Breast Cancer Cells

Purpose

The lung is one of the most common sites of breast cancer metastasis. While metastatic seeding is often accompanied by a dormancy-promoting mesenchymal to epithelial reverting transitions (MErT), we aimed to determine whether lung epithelial cells can impart this phenotype on aggressive breast cancer cells.

Methods

Co-culture experiments of normal lung epithelial cell lines (SAEC, NHBE or BEAS-2B) and breast cancer cell lines (MCF-7 or MDA-MB-231) were conducted. Flow cytometry analysis, immunofluorescence staining for E-cadherin or Ki-67 and senescence associated beta-galactosidase assays assessed breast cancer cell outgrowth and phenotype.

Results

Co-culture of the breast cancer cells with the normal lung cells had different effects on the epithelial and mesenchymal carcinoma cells. The epithelial MCF-7 cells were increased in number but still clustered even if in a slightly more mesenchymal-spindle morphology. On the other hand, the mesenchymal MDA-MB-231 cells survived but did not progressively grow out in co-culture. These aggressive carcinoma cells underwent an epithelial shift as indicated by cuboidal morphology and increased E-cadherin. Disruption of E-cadherin expressed in MDA-MB-231 using shRNA prevented this phenotypic reversion in co-culture. Lung cells limited cancer cell growth kinetics as noted by both (1) some of the cells becoming larger and positive for senescence markers/negative for proliferation marker Ki-67, and (2) Ki-67 positive cells significantly decreasing in MDA-MB-231 and MCF-7 cells after co-culture.

Conclusions

Our data indicate that normal lung epithelial cells can drive an epithelial phenotype and suppress the growth kinetics of breast cancer cells coincident with changing their phenotypes.     

Livin promotes progression of breast cancer through induction of epithelial–mesenchymal transition and activation of AKT signaling

The inhibitor of apoptosis proteins (IAP) are closely correlated with proliferation, apoptosis, motility, and metastasis. Livin is the most recently identified IAP, and its role in breast progression remains unknown. In our study, analyses of 50 patients with breast cancer revealed that the positive expression rate of Livin was higher in breast cancer tissues (62%) relative to that in adjacent (35%) and normal tissues (25%). Livin expression in breast cancer correlated with the clinical stage and axillary lymph node metastasis and could be used as a prognostic marker. Our in vitro experiment revealed that Livin was highly expressed in high-invasive MDA-MB-231 cells as compared to low-invasive cells (MCF-7). Suppression of Livin by short-hairpin RNA reduced the Livin expression of MDA-MB-231 cells and subsequently inhibited tumor cell growth, proliferation, and colony formation and induced tumor cell apoptosis, motility, migration, and invasion. Overexpression of Livin in MCF7 cells resulted in increased migration and invasion capabilities of the cells without affecting proliferation and apoptosis. In addition, epithelial–mesenchymal transition (EMT) was induced by Livin expression in breast cancer cell lines. The high level of phosphorylated AKT in MDA-MB-231 cells was suppressed by Livin knockdown. Further, Livin-induced migration and invasion could be abolished by either the application of the phosphoinositide-3-kinase inhibitor LY294002 or knockdown of AKT expression using small-interfering RNA. In conclusion, Livin serves as an independent prognostic indicator for breast cancer. Livin expression promotes breast cancer metastasis through the activation of AKT signaling and induction of EMT in breast cancer cells both in vitro and in vivo.     

分泌蛋白YKL-40增强肿瘤细胞的上皮间质样转化

目的:分泌糖蛋白YKL-40在多种晚期肿瘤病人的血液中显著升高,提示YKL-40蛋白的血浓度是肿瘤恶变的生物标志物。本课题研究YKL-40重组蛋白和过表达YKL-40肿瘤细胞对肿瘤细胞的上皮间质样转化的作用。方法:构建YKL-40过表达的纤维状乳腺癌细胞系MDA-MB-231和非纤维状结肠癌细胞系HCT-116,观察细胞形态学变化,收集细胞和细胞培养液用于Western Blot(WB)检测YKL-40和上皮间质转化标记蛋白Vimentin和N-cadherin。观察重组蛋白YKL-40对原代MDA-MB-231细胞在无血清条件下的细胞存活影响;另外,用细胞存活试剂盒检测YKL-40过表达HCT-116细胞在无血清的培养液中细胞存活情况。最后,用细胞侵袭试验检测YKL-40过表达MDA-MB-231细胞的侵袭力,并用WB和Zymography来测定细胞分泌MMP9蛋白的表达和酶活性。结果:YKL-40过表达增强MDA-MB-231细胞的形态向上皮间质样转化,并显著提高Vimentin、N-cadherin蛋白的表达,但对HCT-116细胞无法诱导上皮间质样转化。在无血清培养基培养条件下,YKL-40可以增强两种细胞的存活能力,并且YKL-40过表达的MDA-MB-231细胞增强了细胞的侵袭能力,促进了MMP9蛋白表达和蛋白活性。结论:YKL-40可以增加肿瘤细胞的存活力,增强纤维状细胞向上皮间质样转化;并且,YKL-40增加MMP9蛋白表达和活性,增强细胞侵袭力。YKL-40是间质样肿瘤细胞EMT的增强子,此发现为抑制肿瘤恶变提供新靶点。     

Expression of myoferlin in human airway epithelium and its role in cell adhesion and zonula occludens-1 expression

Background

Normal airway epithelial barrier function is maintained by cell-cell contacts which require the translocation of adhesion proteins at the cell surface, through membrane vesicle trafficking and fusion events. Myoferlin and dysferlin, members of the multiple-C2-domain Ferlin superfamily, have been implicated in membrane fusion processes through the induction of membrane curvature. The objectives of this study were to examine the expression of dysferlin and myoferlin within the human airway and determine the roles of these proteins in airway epithelial homeostasis.

Methods

The expression of dysferlin and myoferlin were evaluated in normal human airway sections by immunohistochemistry, and primary human airway epithelial cells and fibroblasts by immuno blot. Localization of dysferlin and myoferlin in epithelial cells were determined using confocal microscopy. Functional outcomes analyzed included cell adhesion, protein expression, and cell detachment following dysferlin and myoferlin siRNA knock-down, using the human bronchial epithelial cell line, 16HBE.

Results

Primary human airway epithelial cells express both dysferlin and myoferlin whereas fibroblasts isolated from bronchi and the parenchyma only express myoferlin. Expression of dysferlin and myoferlin was further localized within the Golgi, cell cytoplasm and plasma membrane of 16HBE cells using confocal micrscopy. Treatment of 16HBE cells with myoferlin siRNA, but not dysferlin siRNA, resulted in a rounded cell morphology and loss of cell adhesion. This cell shedding following myoferlin knockdown was associated with decreased expression of tight junction molecule, zonula occludens-1 (ZO-1) and increased number of cells positive for apoptotic markers Annexin V and propidium iodide. Cell shedding was not associated with release of the innate inflammatory cytokines IL-6 and IL-8.

Conclusions/Significance

This study demonstrates the heterogeneous expression of myoferlin within epithelial cells and fibroblasts of the respiratory airway. The effect of myoferlin on the expression of ZO-1 in airway epithelial cells indicates its role in membrane fusion events that regulate cell detachment and apoptosis within the airway epithelium.     

Down‐regulating Myoferlin inhibits the vasculogenic mimicry of melanoma via decreasing MMP‐2 and inducing mesenchymal‐to‐epithelial transition

Vasculogenic mimicry (VM) constitutes a novel approach for tumour blood supply and contributes to tumour metastasis and poor prognosis in patients with melanoma. Myoferlin (MYOF), a type II membrane protein involved in membrane regeneration and repair, is elevated in several malignant tumours, especially in advanced melanomas. This study aims to investigate the role and mechanism of MYOF in the regulation of VM. VM structures were found in 14 of 52 tested melanoma samples, and high MYOF expression correlated with VM structures. According to Kaplan–Meier survival curves, VM channels and elevated MYOF expression both correlated with poor prognosis in melanoma patients. Down‐regulation of MYOF by siRNA severely impaired the capability of A375 cells to form VM structures in vitro. Further studies demonstrated MYOF knockdown inhibited cell migration and invasion, which is required for VM formation, via decreasing MMP‐2 expression as evidenced by Western blotting, RT‐RCP and ELISA results. SB‐3CT, a specific inhibitor of MMP‐2, showed similar inhibiting effects with siMYOF, further supporting that MYOF down‐regulation inhibits MMP‐2 expression to affect VM formation. Moreover, MYOF knockdown suppress VM formation by A375 cells by inducing mesenchymal‐to‐epithelial transition (MET). After down‐regulating MYOF, focal adhesions were enlarged and A375 cells developed into a clear epithelial morphology. Such cells acquired the expression of E‐cadherin at adherens junctions along with a loss of mesenchymal markers, such as Vimentin and Twist1. In conclusion, MYOF plays an important role in VM and knockdown of MYOF suppresses VM formation via decreasing MMP‐2 and inducing MET in A375 melanoma cells.     

Tumor Suppressor Function of Syk in Human MCF10A In Vitro and Normal Mouse Mammary Epithelium In Vivo

The normal function of Syk in epithelium of the developing or adult breast is not known, however, Syk suppresses tumor growth, invasion, and metastasis in breast cancer cells. Here, we demonstrate that in the mouse mammary gland, loss of one Syk allele profoundly increases proliferation and ductal branching and invasion of epithelial cells through the mammary fat pad during puberty. Mammary carcinomas develop by one year. Syk also suppresses proliferation and invasion in vitro. siRNA or shRNA knockdown of Syk in MCF10A breast epithelial cells dramatically increased proliferation, anchorage independent growth, cellular motility, and invasion, with formation of functional, extracellular matrix-degrading invadopodia. Morphological and gene microarray analysis following Syk knockdown revealed a loss of luminal and differentiated epithelial features with epithelial to mesenchymal transition and a gain in invadopodial cell surface markers CD44, CD49F, and MMP14. These results support the role of Syk in limiting proliferation and invasion of epithelial cells during normal morphogenesis, and emphasize the critical role of Syk as a tumor suppressor for breast cancer. The question of breast cancer risk following systemic anti-Syk therapy is raised since only partial loss of Syk was sufficient to induce mammary carcinomas.     

Progranulin stimulated by LPA promotes the migration of aggressive breast cancer cells

Activator and inhibitor roles for the 88-kDa-secreted glycoprotein progranulin (PGRN) have been demonstrated in ovarian cancer cells. Here, we investigated the effects of PGRN in breast cancer migration. Testing MCF7, MDA-MB-453, and MDA-MB-231 human breast cancer cells and the MCF10A breast epithelial cell line, we demonstrate that LPA-induced PGRN stimulation led to a significant increase in cell invasion of MDA-MB-453 and MDA-MB-231 cells only (p<0.05). Moreover, incubation with an anti-PGRN antibody, an inhibitor of the ERK pathway (PD98059) or both in combination inhibited the ability of MDA-MB-231 cells to invade. Furthermore, the expression of focal adhesion kinases promoted by LPA-induced PGRN was also inhibited by PD98059 alone or in combination with an anti-PGRN antibody (p<0.05). Taken together, these results suggest that the LPA activation of PGRN involving the ERK pathway is critical to promote MDA-MB-231 breast cancer cell invasion.     

Myoferlin,a multifunctional protein in normal cells,has novel and key roles in various cancers

Myoferlin, a protein of the ferlin family, has seven C2 domains and exhibits activity in some cells, including myoblasts and endothelial cells. Recently, myoferlin was identified as a promising target and biomarker in non‐small‐cell lung cancer, breast cancer, pancreatic adenocarcinoma, hepatocellular carcinoma, colon cancer, melanoma, oropharyngeal squamous cell carcinoma, head and neck squamous cell carcinoma, clear cell renal cell carcinoma and endometrioid carcinoma. This evidence indicated that myoferlin was involved in the proliferation, invasion and migration of tumour cells, the mechanism of which mainly included promoting angiogenesis, vasculogenic mimicry, energy metabolism reprogramming, epithelial‐mesenchymal transition and modulating exosomes. The roles of myoferlin in both normal cells and cancer cells are of great significance to provide novel and efficient methods of tumour treatment. In this review, we summarize recent studies and findings of myoferlin and suggest that myoferlin is a novel potential candidate for clinical diagnosis and targeted cancer therapy.     

Selective Androgen Receptor Modulators (SARMs) Negatively Regulate Triple-Negative Breast Cancer Growth and Epithelial:Mesenchymal Stem Cell Signaling

Introduction

The androgen receptor (AR) is the most highly expressed steroid receptor in breast cancer with 75–95% of estrogen receptor (ER)-positive and 40–70% of ER-negative breast cancers expressing AR. Though historically breast cancers were treated with steroidal androgens, their use fell from favor because of their virilizing side effects and the emergence of tamoxifen. Nonsteroidal, tissue selective androgen receptor modulators (SARMs) may provide a novel targeted approach to exploit the therapeutic benefits of androgen therapy in breast cancer.

Materials and Methods

Since MDA-MB-453 triple-negative breast cancer cells express mutated AR, PTEN, and p53, MDA-MB-231 triple-negative breast cancer cells stably expressing wildtype AR (MDA-MB-231-AR) were used to evaluate the in vitro and in vivo anti-proliferative effects of SARMs. Microarray analysis and epithelial:mesenchymal stem cell (MSC) co-culture signaling studies were performed to understand the mechanisms of action.

Results

Dihydrotestosterone and SARMs, but not bicalutamide, inhibited the proliferation of MDA-MB-231-AR. The SARMs reduced the MDA-MB-231-AR tumor growth and tumor weight by greater than 90%, compared to vehicle-treated tumors. SARM treatment inhibited the intratumoral expression of genes and pathways that promote breast cancer development through its actions on the AR. SARM treatment also inhibited the metastasis-promoting paracrine factors, IL6 and MMP13, and subsequent migration and invasion of epithelial:MSC co-cultures.

Conclusion

1. AR stimulation inhibits paracrine factors that are important for MSC interactions and breast cancer invasion and metastasis. 2. SARMs may provide promise as novel targeted therapies to treat AR-positive triple-negative breast cancer.     

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。     

Progranulin Stimulated by LPA Promotes the Migration of Aggressive Breast Cancer Cells

Abstract

Activator and inhibitor roles for the 88-kDa-secreted glycoprotein progranulin (PGRN) have been demonstrated in ovarian cancer cells. Here, we investigated the effects of PGRN in breast cancer migration. Testing MCF7, MDA-MB-453, and MDA-MB-231 human breast cancer cells and the MCF10A breast epithelial cell line, we demonstrate that LPA-induced PGRN stimulation led to a significant increase in cell invasion of MDA-MB-453 and MDA-MB-231 cells only (p < 0.05). Moreover, incubation with an anti-PGRN antibody, an inhibitor of the ERK pathway (PD98059) or both in combination inhibited the ability of MDA-MB-231 cells to invade. Furthermore, the expression of focal adhesion kinases promoted by LPA-induced PGRN was also inhibited by PD98059 alone or in combination with an anti-PGRN antibody (p < 0.05). Taken together, these results suggest that the LPA activation of PGRN involving the ERK pathway is critical to promote MDA-MB-231 breast cancer cell invasion.     

沉默ST3Gal III抑制MDA-MB-231细胞黏附和侵袭能力

我们前期研究表明α2,3-唾液酸水平与乳腺癌侵袭转移密切相关。人α2,3-唾液酸转移酶（ST3Gal Ⅲ）可催化合成细胞表面的α2,3-唾液酸,并在乳腺癌组织中高表达,此酶活性与肿瘤转移潜能密切相关,但其机制尚未阐明。本研究中我们将继续探讨ST3Gal Ⅲ在对乳腺癌转移关键步骤粘附和侵袭中的作用。构建特异靶向ST3Gal Ⅲ的短发夹RNA（shRNA）序列的慢病毒载体,采用细胞转染沉默乳腺癌MDA-MB-231细胞的ST3Gal Ⅲ,经实时定量PCR及Western印迹检测转染后细胞ST3Gal Ⅲ mRNA及蛋白表达,验证构建了稳定下调ST3Gal Ⅲ表达的两个细胞克隆,分别记作shRNA-2、shRNA-4。细胞表面α2,3-唾液酸是ST3Gal Ⅲ下游产物,可代表酶活性。流式细胞术分析结果证实,shRNA-2、shRNA-4细胞表面α2,3-唾液酸的含量显著降低（P<0.05）。细胞黏附、细胞迁移及侵袭能力等功能学检测结果表明,shRNA细胞黏附能力及侵袭能力明显降低（P<0.05）。β1整合素表达与肿瘤侵袭能力获取密切相关。本研究中,沉默ST3Gal Ⅲ可抑制β1整合素表达（P<0.05）。这些结果提示,ST3Gal Ⅲ在乳腺癌转移关键步骤黏附和侵袭中具有重要作用,沉默ST3Gal Ⅲ抑制MDA MB-231细胞黏附和侵袭能力,其作用机制可能是通过下调β1整合素表达。此研究从新的视角认识了乳腺癌转移的机制,并可能提供乳腺癌转移治疗的新靶点。     

Production of bioengineered cancer tissue constructs in vitro: epithelium-mesenchyme heterotypic interactions

A few models have been established to study cancer cells in vitro. However, the cellular interactions have rarely been studied specifically using bioengineered cancer constructs combining human carcinoma cells and tumor-associated fibroblasts. We developed an in vitro model of tridimensional bioengineered cancer tissue constructs (bCTC) by seeding mammary epithelial cancer cells or normal keratinocytes over a mesenchymal layer containing tumor-derived fibroblastic cells or normal skin fibroblasts. After the introduction of epithelial cells, each construct was cultured for another 10 d. Histologic analyses showed that carcinoma cell lines could invade the subjacent mesenchymal layer and that the capacity to migrate was related to the invasive potential of cancer cells and the type of fibroblasts used, while noninvasive populations did not. Of the tested epithelial cells, MDA-MB-231 and, to a lesser degree, HDQ-P1 cell lines were invasive, and the invasion was deeper into the mesenchymal component containing tumor-derived fibroblasts. However, with normal skin fibroblasts, the mesenchymal layer was degraded twice faster than with tumor-derived fibroblastic cells. MDA-MB-231 cells and normal keratinocytes induced the highest level of gelatinase B, and the level was lowest with the MCF-7 cell line. The activated form of gelatinase B was, however, induced to the highest levels in the keratinocyte-seeded bCTC containing tumor-derived but not normal fibroblasts. MDA-MB-231 was the only epithelial cancer cell line whose activity of gelatinase A was reduced when cocultured with tumor-derived fibroblasts but not under normal fibroblast stimulation. Finally, a 50/48-kDa gelatinase band has been observed in bCTCs with noninvasive epithelial cells only. Our study demonstrates the selective secretion of gelatinases according to the phenotype of the cells seeded in the various bCTCs.     

Toll-like receptor 9 mediates CpG oligonucleotide-induced cellular invasion

Toll-like receptor 9 (TLR9) belongs to the innate immune system and recognizes microbial and vertebrate DNA. We showed previously that treatment with the TLR9-agonistic ODN M362 (a CpG sequence containing oligonucleotide) induces matrix metalloproteinase-13-mediated invasion in TLR9-expressing human cancer cell lines. Here, we further characterized the role of the TLR9 pathway in this process. We show that CpG oligonucleotides induce invasion in macrophages from wild-type C57/B6 and MyD88 knockout mice and in human MDA-MB-231 breast cancer cells lacking MyD88 expression. This effect was significantly inhibited in macrophages from TLR9 knockout mice and in human MDA-MB-231 breast cancer cells stably expressing TLR9 small interfering RNA or dominant-negative tumor necrosis factor receptor-associated factor 6 (TRAF6). Sequence modifications to the CpG oligonucleotides that targeted the stem loop and other secondary structures were shown to influence the invasion-inducing effect in MDA-MB-231 cells. In contrast, methylation of the cytosine residues of the parent CpG oligonucleotide did not affect the TLR9-mediated invasion compared with the unmethylated parent CpG oligonucleotide. Finally, expression of TLR9 was studied in clinical breast cancer samples and normal breast epithelium with immunohistochemistry. TLR9 staining localized in epithelial cells in both cancer and normal samples. The mean TLR9 staining intensity was significantly increased in the breast cancer cells compared with normal breast epithelial cells. In conclusion, our results suggest that TLR9 expression is increased in breast cancer and CpG oligonucleotide-induced cellular invasion is mediated via TLR9 and TRAF6, independent of MyD88. Further, our findings suggest that the structure and/or stability of DNA may influence the induction of TLR9-mediated invasion in breast cancer.     

The monoamine oxidase-A inhibitor clorgyline promotes a mesenchymal-to-epithelial transition in the MDA-MB-231 breast cancer cell line

Monoamine oxidase-A (MAO-A) dysfunction has been historically associated with depression. Recently, depression as well as altered MAO-A expression have both been associated with a poor prognosis in cancers, although the mechanism involved remains ambiguous. For example, MAO-A mRNA is repressed across cancers, yet MAO-A protein and levels of serotonin, a substrate of MAO-A implicated in depression, are paradoxically increased in malignancies, including breast cancer.The effect of clorgyline (CLG), a selective inhibitor of MAO-A, on malignant behaviour, expression of transitional markers, and biochemical correlates was examined in two human breast carcinoma cell lines, i.e. the epithelial, oestrogen receptor (ER)-positive MCF-7 cell line and the post-EMT (mesenchymal), ER-negative MDA-MB-231 cell line.CLG exerted little effect on malignant behaviour in MCF-7 cells, but inhibited proliferation and anchorage-independent growth, and increased invasiveness and active migration of MDA-MB-231 cells. CLG induced the expression of the mesenchymal marker vimentin in MCF-7 cells, but not in MDA-MB-231 cells. In contrast, CLG induced the epithelial protein marker E-cadherin in both cell lines, with a more robust effect in MDA-MB-231 cells (where a nuclear E-cadherin signal was also detected). This effect appears to be independent of any canonical Snai1-mediated regulation of E-cadherin mRNA expression. CLG interfered with the β-catenin/[phospho]GSK-3β complex as well as the E-cadherin/β-catenin complex in both cell lines cells, but, again, the effect was more robust in MDA-MB-231 cells. Parallel studies revealed a general lack of effect of CLG on the ER-negative, epithelial Au565 breast cancer cell line. Thus, any effect of CLG on metastatic behaviours appears to rely on the cell's EMT status rather than on the cell's ER status.These data suggest that inactivation of MAO-A triggers a mesenchymal-to-epithelial transition in MDA-MB-231 cells via a non-canonical mechanism. This potentially implicates an MAO-A-sensitive step in advanced breast cancer and should be borne in mind when considering pharmacological treatment options for co-morbid depression in breast cancer patients.     

Small molecule inhibition of arylamine N-acetyltransferase Type I inhibits proliferation and invasiveness of MDA-MB-231 breast cancer cells

Arylamine N-acetyltransferase 1 is a phase II metabolizing enzyme that has been associated with certain breast cancer subtypes. While it has been linked to breast cancer risk because of its role in the metabolic activation and detoxification of carcinogens, recent studies have suggested it may be important in cell growth and survival. To address the possible importance of NAT1 in breast cancer, we have used a novel small molecule inhibitor (Rhod-o-hp) of the enzyme to examine growth and invasion of the breast adenocarcinoma line MDA-MB-231. The inhibitor significantly reduced cell growth by increasing the percent of cells in G2/M phase of the cell cycle. Rhod-o-hp also reduced the ability of the MDA-MB-231 cells to grow in soft agar. Using an in vitro invasion assay, the inhibitor significantly reduced the invasiveness of the cells. To test whether this effect was due to inhibition of NAT1, the enzyme was knocked down using a lentivirus-based shRNA approach and invasion potential was significantly reduced. Taken together, the results of this study demonstrate that NAT1 activity may be important in breast cancer growth and metastasis. The study suggests that NAT1 is a novel target for breast cancer treatment.     

Promotion of experimental thrombus formation by the procoagulant activity of breast cancer cells

The routine observation of tumor emboli in the peripheral blood of patients with carcinomas raises questions about the clinical relevance of these circulating tumor cells. Thrombosis is a common clinical manifestation of cancer, and circulating tumor cells may play a pathogenetic role in this process. The presence of coagulation-associated molecules on cancer cells has been described, but the mechanisms by which circulating tumor cells augment or alter coagulation remains unclear. In this study we utilized suspensions of a metastatic adenocarcinoma cell line, MDA-MB-231, and a non-metastatic breast epithelial cell line, MCF-10A, as models of circulating tumor cells to determine the thrombogenic activity of these blood-foreign cells. In human plasma, both metastatic MDA-MB-231 cells and non-metastatic MCF-10A cells significantly enhanced clotting kinetics. The effect of MDA-MB-231 and MCF-10A cells on clotting times was cell number-dependent and inhibited by a neutralizing antibody to tissue factor (TF) as well as inhibitors of activated factor X and thrombin. Using fluorescence microscopy, we found that both MDA-MB-231 and MCF-10A cells supported the binding of fluorescently labeled thrombin. Furthermore, in a model of thrombus formation under pressure-driven flow, MDA-MB-231 and MCF-10A cells significantly decreased the time to occlusion. Our findings indicate that the presence of breast epithelial cells in blood can stimulate coagulation in a TF-dependent manner, suggesting that tumor cells that enter the circulation may promote the formation of occlusive thrombi under shear flow conditions.