Cancer Cell Expression of Autotaxin Controls Bone Metastasis Formation in Mouse through Lysophosphatidic Acid-Dependent Activation of Osteoclasts

Background

Bone metastases are highly frequent complications of breast cancers. Current bone metastasis treatments using powerful anti-resorbtive agents are only palliative indicating that factors independent of bone resorption control bone metastasis progression. Autotaxin (ATX/NPP2) is a secreted protein with both oncogenic and pro-metastatic properties. Through its lysosphospholipase D (lysoPLD) activity, ATX controls the level of lysophosphatidic acid (LPA) in the blood. Platelet-derived LPA promotes the progression of osteolytic bone metastases of breast cancer cells. We asked whether ATX was involved in the bone metastasis process. We characterized the role of ATX in osteolytic bone metastasis formation by using genetically modified breast cancer cells exploited on different osteolytic bone metastasis mouse models.

Methodology/Principal Findings

Intravenous injection of human breast cancer MDA-B02 cells with forced expression of ATX (MDA-B02/ATX) to inmmunodeficiency BALB/C nude mice enhanced osteolytic bone metastasis formation, as judged by increased bone loss, tumor burden, and a higher number of active osteoclasts at the metastatic site. Mouse breast cancer 4T1 cells induced the formation of osteolytic bone metastases after intracardiac injection in immunocompetent BALB/C mice. These cells expressed active ATX and silencing ATX expression inhibited the extent of osteolytic bone lesions and decreased the number of active osteoclasts at the bone metastatic site. In vitro, osteoclast differentiation was enhanced in presence of MDA-B02/ATX cell conditioned media or recombinant autotaxin that was blocked by the autotaxin inhibitor vpc8a202. In vitro, addition of LPA to active charcoal-treated serum restored the capacity of the serum to support RANK-L/MCSF-induced osteoclastogenesis.

Conclusion/Significance

Expression of autotaxin by cancer cells controls osteolytic bone metastasis formation. This work demonstrates a new role for LPA as a factor that stimulates directly cancer growth and metastasis, and osteoclast differentiation. Therefore, targeting the autotaxin/LPA track emerges as a potential new therapeutic approach to improve the outcome of patients with bone metastases.     

乳腺癌细胞迁移的调节与转移

细胞迁移是乳腺癌侵袭和转移中的关键步骤之一.癌细胞在迁移过程中主要受到Rho GTPases的调节,发生肌动蛋白骨架重组,获得定向迁移的能力;高迁移能力的癌细胞通过与胞外基质成分相互作用,为迁移创造合适的微环境;最后迁移的癌细胞在靶器官的趋化作用下在特定部位驻足生长,这些环节共同作用导致乳腺癌转移.研究细胞迁移复杂的分子机制将为控制乳腺癌转移提供新的策略.     

Chemoattractant Signaling between Tumor Cells and Macrophages Regulates Cancer Cell Migration,Metastasis and Neovascularization

Tumor-associated macrophages are known to influence cancer progression by modulation of immune function, angiogenesis, and cell metastasis, however, little is known about the chemokine signaling networks that regulate this process. Utilizing CT26 colon cancer cells and RAW 264.7 macrophages as a model cellular system, we demonstrate that treatment of CT26 cells with RAW 264.7 conditioned medium induces cell migration, invasion and metastasis. Inflammatory gene microarray analysis indicated CT26-stimulated RAW 264.7 macrophages upregulate SDF-1α and VEGF, and that these cytokines contribute to CT26 migration in vitro. RAW 264.7 macrophages also showed a robust chemotactic response towards CT26-derived chemokines. In particular, microarray analysis and functional testing revealed CSF-1 as the major chemoattractant for RAW 264.7 macrophages. Interestingly, in the chick CAM model of cancer progression, RAW 264.7 macrophages localized specifically to the tumor periphery where they were found to increase CT26 tumor growth, microvascular density, vascular disruption, and lung metastasis, suggesting these cells home to actively invading areas of the tumor, but not the hypoxic core of the tumor mass. In support of these findings, hypoxic conditions down regulated CSF-1 production in several tumor cell lines and decreased RAW 264.7 macrophage migration in vitro. Together our findings suggest a model where normoxic tumor cells release CSF-1 to recruit macrophages to the tumor periphery where they secrete motility and angiogenic factors that facilitate tumor cell invasion and metastasis.     

具有乳腺导管再生能力的小鼠乳腺原基细胞群黏附因子的表达特点

乳腺干细胞是研究器官形成、细胞增殖、分化、生存和凋亡等信号通路的理想模型,而近来的研究发现许多成体干细胞的特异性表面标记都与细胞黏附分子(celladhesion molecule,CAM)家族相关.因此,研究胚胎期乳腺干/祖细胞群的黏附分子基因表达特点,对于纯化和鉴定胚胎期乳腺干/祖细胞具有重要指导意义.用成年小鼠乳腺上皮干细胞的标记CD24和CD49f来分选小鼠胚胎期14天乳腺原基细胞群,发现CD24+和CD49f+双阳性的乳腺原基细胞包含两个细胞群:CD24hiCD49f+细胞群和CD24medCD49f+细胞群.它们占乳腺原基总细胞的比例分别为16%和47%.在随后的细胞培养实验和体内移植再生实验中发现,CD24medCD49f+细胞群可以贴壁,而且具有再生乳腺导管的能力,相反,CD24hiCD49f+细胞群既不能贴壁也不具有移植再生能力.这些结果表明,这两个细胞群分别代表不同的细胞类型,而CD24medCD49f+细胞群有可能包含具有自我更新能力的乳腺原基干/祖细胞.挑选了可能与乳腺相关的19个黏附分子,并对这两群细胞进行了定量PCR检测.结果表明,具有乳腺导管重建能力的CD24medCD49f+细...     

G1 Cell Cycle Regulatory Proteins in Chemically-Induced Rat Mammary Adenocarcinomas In Vivo and Tumor Promotion-Sensitive, -Resistant and Transformed Mouse Epidermal Cells In Vitro

Tumor promotion is characterized by selective proliferation of initiated cells resulting in their clonal expansion. Cyclin D1 is frequently upregulated in this process, but its expression does not necessarily correlate positively with cyclin A. In the present article, expression of G₁ cell cycle regulatory proteins was systematically analyzed using two models of carcinogenesis: (a) N-methyl-N-nitrosourea (MNU)-induced rat mammary adenocarcinomas and normal rat mammary epithelial cells in vivo and (b) promotion- sensitive, -resistant, and transformed JB6 mouse epidermal cells in vitro. The results of this analysis revealed that p27Kip1 negatively correlated with cyclin D1. In addition, there were two types of correlations between p27Kip1 and cyclin A. First, p27Kip1 negatively correlated with cyclin A (type-I correlation). This scenario was observed in normal rat mammary epithelial cells in vivo and promotion-sensitive (P+) JB6 mouse epidermal cells, stimulated with phorbol ester (TPA) in vitro. Second, p27Kip1 positively correlated with cyclin A (type-II correlation). This correlation was observed in MNU-induced rat mammary adenocarcinomas in vivo and TPA-stimulated (P+) JB6 cells, treated with retinoic acid in vitro.     

前列腺癌骨转移：肿瘤细胞与骨微环境之间的相互作用

  肿瘤转移包括一系列复杂的过程,主要涉及肿瘤细胞由原发部位脱离开始,到肿瘤细胞在其它转移部位——比如骨骼,生长增殖的多个关键性步骤.“种子和土壤学说”预示骨微环境中表达许多因子,吸引多种肿瘤细胞的迁移以及促进肿瘤的增殖.通过肿瘤细胞与其所处生长环境中的双向和动态的作用,促进肿瘤在骨骼中的发展.因此,骨微环境中产生的因子对肿瘤骨转移具有重要意义.本综述以前列腺癌为例,总结了肿瘤转移的机理研究概况,特别强调了目前有关肿瘤细胞与骨微环境之间相互作用研究的重要性,并提出了未来的研究方向     

Expression and Localization of GEN1 in Mouse Mammary Epithelial Cells

GEN1, a Holliday junction resolvase, is involved in homologous repair of DNA double strand break and in maintaining centrosome integrity. Although GEN1 mutants have been reported in breast cancer patients and cell lines, little is currently known about the functions of GEN1 in the development and oncogenic transformation of mammary gland. In the present study, we demonstrate that GEN1 expression is correlated with mammary epithelial cell proliferation, differentiation in various physiological stages as well as casein. By immunofluorescence analysis, the centrosomal association of GEN1 is confirmed in mammary epithelial cells. Additionally, GEN1 is likely involved in DNA damage response of breast cancer cell lines. These results suggest that GEN1 may play an important role in the development of mammary gland; its response upon DNA damage indicates that GEN1 gene alteration may contribute to breast cancer formation.     

Isolation of an Insulin-Responsive Preadipose Cell Line and a Mammary Tumor Virus-Producing, Dome-Forming Epithelial Cell Line from a Mouse Mammary Tumor

Two functional tissue culture cell lines, MTD and MTF cell lines, have been isolated from a mouse mammary tumor. MTD cells are epithelial and retain the ability to transport fluid leading to the formation of three-dimensional fluid-filled multicellular structures called "domes" or "hemicysts". Another property of MTD cells is the production of murine mammary tumor virus (MTV). Release of MTV into the culture medium was verified by immunological, electrophoretic and enzymatic analyses. Addition of dexamethasone in the culture medium enhanced both the formation of domes and the production of MTV. Thus, MTD cells retain the morphological and functional properties of the original mammary tumor cells.
MTF cells show the fibroblastic morphology in subconfluent cultures. After reaching confluence, however, these cells gradually accumulated triglycerides in the cytoplasm and eventually assumed the morphology of fat cells. This adipose conversion was greatly enhanced by the presence of insulin in the culture medium. The morphological resemblance of adipose-converted MTF cells to the mammary fat cells suggests that the MTF cell line was derived from the mammary fat pad stroma. These functional cell lines will be useful to study cell differentiation as well as cell-to-cell interactions in the mammary gland.     

细胞膜表面糖链结构与肿瘤转移的关系

连接在天冬酰胺上的N-糖链,连接在丝氨酸和苏氨酸上的O-糖链,连接在丝氨酸上的糖胺聚糖,连接在脂类物质上的糖脂等糖基化修饰在真核细胞表面普遍存在,并调节了细胞的各种功能,它们不仅参与生命活动中正常的生理生化活动,而且于疾病的发生发展密不可分。肿瘤细胞表面糖基异常化,如糖链在表达水平上的差异以及特殊糖链结构的出现,均与肿瘤细胞的侵袭和转移有密切关系。本综述主要介绍了肿瘤细胞糖基化的改变:一些主要的糖基化结构如β1,6分支和唾液酸的表达的增加会使某些肿瘤细胞的迁移能力增强。同时对引起这种变化的作用机制进行了介绍,提出一些潜在的抗肿瘤研究靶点,为肿瘤糖生物学的深入研究提供参考。     

Establishment of a C3Hf Mammary Tumor Cell Line Expressing Endogenous Mouse Mammary Tumor Virus: Antigenic and Genetic Relationships of This Virus with Highly Oncogenic Mouse Mammary Tumor Viruses

A single-cell clone of C3Hf mammary tumor cells (clone 14) was developed into a continuous cell line expressing high levels of endogenous mouse mammary tumor virus (MMTV) with less than 0.1% murine leukemia virus expression. Comparison of the C3Hf MMTV protein profile on sodium dodecyl sulfatepolyacrylamide gel electrophoresis with that of C3H MMTV revealed that the protein content of the two viruses was quite similar. However, oligonucleotide fingerprints obtained of MMTV 70S RNA revealed that approximately 20% of the large oligonucleotides examined were unique to each virus. The oligonucleotide fingerprint indicated that although the viruses were similar, they differed in their genetic content. The differences in the two viruses extended to immunological differences in the major envelope glycoprotein, gp52. C3Hf MMTV competed only partially in a homologous radioimmunoassay for gp52 of C3H MMTV, whereas C3H MMTV gave complete competition, indicating that gp52 of C3H MMTV contained type-specific determinants not present on gp52 of C3Hf MMTV. Comparison of C3Hf MMTV with highly oncogenic C3H, GR, and RIII MMTVs in a homologous C3H MMTV gp52 assay gave two patterns of reactivity: complete competition by GR and C3H MMTV and incomplete competition by C3Hf and RIII MMTV. Absorption of anti-C3H MMTV serum by either C3Hf MMTV or RIII MMTV removed all antibodies against both viruses but not against GR and C3H MMTVs. These results indicate that C3H and GR MMTVs are more closely related to each other than to RIII and C3Hf MMTVs.     

Multimodality Imaging of Tumor and Bone Response in a Mouse Model of Bony Metastasis

Cancer drug development generally performs in vivo evaluation of treatment effects that have traditionally relied on detection of morphologic changes. The emergence of new targeted therapies, which may not result in gross morphologic changes, has spurred investigation into more specific imaging methods to quantify response, such as targeted fluorescent probes and bioluminescent cells. The present study investigated tissue response to docetaxel or zoledronic acid (ZA) in a mouse model of bony metastasis. Intratibial implantations of breast cancer cells (MDA-MB-231) were monitored throughout this study using several modalities: molecular resonance imaging (MRI) tumor volume and apparent diffusion coefficient (ADC), micro-computed tomography (µCT) bone volume, bioluminescence imaging (BLI) reporting cancer cell apoptosis, and fluorescence using Osteosense 800 and CatK 680-FAST. Docetaxel treatment resulted in tumor cell kill reflected by ADC and BLI increases and tumor volume reduction, with delayed bone recovery seen in µCT prefaced by increased osteoblastic activity (Osteosense 800). In contrast, the ZA treatment group produced similar values in MRI, BLI, and Osteosense 800 fluorescence imaging readouts when compared to controls. However, µCT bone volume increased significantly by the first week post-treatment and the CatK 680-FAST signal was slightly diminished by 4 weeks following ZA treatment. Multimodality imaging provides a more comprehensive tool for new drug evaluation and efficacy screening through identification of morphology as well as function and apoptotic signaling.     

The Rac Inhibitor EHop-016 Inhibits Mammary Tumor Growth and Metastasis in a Nude Mouse Model

Metastatic disease still lacks effective treatments, and remains the primary cause of cancer mortality. Therefore, there is a critical need to develop better strategies to inhibit metastatic cancer. The Rho family GTPase Rac is an ideal target for anti-metastatic cancer therapy, because Rac is a key molecular switch that is activated by a myriad of cell surface receptors to promote cancer cell migration/invasion and survival. Previously, we reported the design and development of EHop-016, a small molecule compound, which inhibits Rac activity of metastatic cancer cells with an IC₅₀ of 1 μM. EHop-016 also inhibits the activity of the Rac downstream effector p21-activated kinase (PAK), lamellipodia extension, and cell migration in metastatic cancer cells. Herein, we tested the efficacy of EHop-016 in a nude mouse model of experimental metastasis, where EHop-016 administration at 25 mg/kg body weight (BW) significantly reduced mammary fat pad tumor growth, metastasis, and angiogenesis. As quantified by UPLC MS/MS, EHop-016 was detectable in the plasma of nude mice at 17 to 23 ng/ml levels at 12 h following intraperitoneal (i.p.) administration of 10 to 25 mg/kg BW EHop-016. The EHop-016 mediated inhibition of angiogenesis In Vivo was confirmed by immunohistochemistry of excised tumors and by In Vitro tube formation assays of endothelial cells. Moreover, EHop-016 affected cell viability by down-regulating Akt and Jun kinase activities and c-Myc and Cyclin D expression, as well as increasing caspase 3/7 activities in metastatic cancer cells. In conclusion, EHop-016 has potential as an anticancer compound to block cancer progression via multiple Rac-directed mechanisms.     

Pleiotrophin (PTN) Expression and Function and in the Mouse Mammary Gland and Mammary Epithelial Cells

Expression of the heparin-binding growth factor, pleiotrophin (PTN) in the mammary gland has been reported but its function during mammary gland development is not known. We examined the expression of PTN and its receptor ALK (Anaplastic Lymphoma Kinase) at various stages of mouse mammary gland development and found that their expression in epithelial cells is regulated in parallel during pregnancy. A 30-fold downregulation of PTN mRNA expression was observed during mid-pregnancy when the mammary gland undergoes lobular-alveolar differentiation. After weaning of pups, PTN expression was restored although baseline expression of PTN was reduced significantly in mammary glands of mice that had undergone multiple pregnancies. We found PTN expressed in epithelial cells of the mammary gland and thus used a monoclonal anti-PTN blocking antibody to elucidate its function in cultured mammary epithelial cells (MECs) as well as during gland development. Real-time impedance monitoring of MECs growth, migration and invasion during anti-PTN blocking antibody treatment showed that MECs motility and invasion but not proliferation depend on the activity of endogenous PTN. Increased number of mammospheres with laminin deposition after anti-PTN blocking antibody treatment of MECs in 3D culture and expression of progenitor markers suggest that the endogenously expressed PTN inhibits the expansion and differentiation of epithelial progenitor cells by disrupting cell-matrix adhesion. In vivo, PTN activity was found to inhibit ductal outgrowth and branching via the inhibition of phospho ERK1/2 signaling in the mammary epithelial cells. We conclude that PTN delays the maturation of the mammary gland by maintaining mammary epithelial cells in a progenitor phenotype and by inhibiting their differentiation during mammary gland development.     

Down Regulation of T Cell Receptor Expression in COPD Pulmonary CD8 Cells

CD8 cells may contribute towards an autoimmune process in COPD. Down regulation of T cell receptor (TCR) signalling molecules occurs in autoimmune diseases with consequent T cell dysfunction. We hypothesise that TCR signalling is abnormal in COPD pulmonary CD8 cells. Micro-array gene expression analysis of blood and pulmonary COPD CD8 samples was performed and compared to pulmonary CD8 cells from smoker controls (S). We focused on the TCR signalling pathway, with validation of key findings using polymerase chain reaction and immunofluorescence. TCR signalling molecules in COPD pulmonary CD8 cells were down regulated compared to blood CD8 cells (CD247: fold change (FC) −2.43, Q = 0.001; LCK: FC −2.25, Q = 0.01). Micro-array analysis revealed no significant differences between COPD and S pulmonary CD8 cells. However, PCR revealed significantly lower gene expression levels of CD247 (FC −1.79, p = 0.04) and LCK (FC −1.77, p = 0.01) in COPD compared to S pulmonary CD8 cells. CD247 down regulation in COPD CD8 cells was confirmed by immunofluorescent staining of bronchoalveolar lavage cells: Significantly fewer COPD CD8 cells co-expressed CD247 compared to healthy non-smoker CD8 cells (mean 88.9 vs 75.2%, p<0.05) There is down regulation of TCR signalling molecules in COPD pulmonary CD8 cells. This may cause T cell dysfunction.