Elevated Levels of Annexin I Protein in vitro and in vivo in Rat and Human Mammary Adenocarcinoma

Annexin I is a phospholipid and actin binding protein which may play a role in signal transduction to the cytoskeleton. Previous work reported the differential expression of annexin I mRNA among rat adenocarcinoma cell lines of various metastatic potential (MTLn3, MTLn2, MTC.4: highest to lowest, respectively) (Pencil et al. 1993, Breast Cancer Res Treat, 25, 165–74). This relationship has been extended to the protein level in in vitro cultures using Western blotting and flow cytometry. Annexin I protein levels in MTLn3 cells are 3- to 5-fold higher than in MTC.4 cells. The weakly metastatic cell line MTLn2 shows levels 1.5- to 2.5-fold higher than MTC.4. In vivo tumors were produced by injecting 1 × 10⁶ cells into mammary fat pads of syngeneic rats and necropsies were performed 40 days later. Semiquantitative immunohistochemical color image analysis was performed using a polyclonal rat annexin I specific antibody. Annexin I protein expression was highest in lung metastases of MTLn3, at 8-fold the levels observed in the MTC.4 primary tumors. MTLn3 cells in the primary tumor had an annexin I specific optical density 3-fold higher than that of cells in the MTC.4 primary tumor. MTLn2 primary tumors had an annexin I specific optical density 1.5- fold higher than MTC.4. A proportion of human mammary adenocarcinomas also have positive annexin I immunoreactivity, often with more uniform annexin I staining in the lymph node metastases. These results suggest that there may be survival advantages for nascent metastatic cells with high annexin I levels This revised version was published online in August 2006 with corrections to the Cover Date.     

Effects of RA 233 treatment on the adhesive,invasive and metastatic properties of 13762NF rat mammary tumor cells

The pyrimido-pyrimidine analogue RA 233 has pleiotropic and differential effects on cultured tumor cell clones isolated from the 13762NF rat mammary adenocarcinoma. A nonresponsive clone of low metastatic potential (MTC) was not modified in its cell fragility or invasive, adhesive or lung-colonizing properties by RA 233 treatment. In contrast, a drug-responsive clone of high metastatic potential (MTLn3) was rendered less invasive and its cell fragility was decreased with RA 233 treatment, although its adhesiveness to lung microvascular endothelial cells and subendothelial matrix was unaffected by RA 233. Lung colonization of intravenously injected MTLn3 cells in syngeneic rats was significantly increased by RA 233 treatment, whereas spontaneous metastasis from the mammary fat pad to lung sites was decreased, although this decrease was not statistically significant.     

Proton NMR examination of tumor cells of high or low metastatic potential

Three rat 13762NF mammary adenocarcinoma clones and cell lines of different metastatic potentials (MTLn3, MTC, and MTPa) were studied for their proton nuclear magnetic resonance spectral characteristics as intact cells in vitro and after chloroform/methanol, neuraminidase, or ethanol treatments. The intact-cell spectral characteristics of the highly metastatic tumor cell clone MTLn3 were clearly distinguished from the less metastatic clone MTC or the parental MTPa cell line on the basis of spectral peaks in the range of 0·9 to 1·45 p.p.m. broad peaks near 2·0 p.p.m., and peaks in the range of 2·75 to 3·2 p.p.m. Glycoproteins are among the molecules known to have resonances in these upfield spectral regions, and these tumor cell subpopulations have previously been shown to possess characteristic quantitative differences in cell surface, metastasis-associated glycoproteins. Treatment of the cells with neuraminidase or ethanol, or extraction with chloroform/methanol increased spectral detail and also revealed characteristic differences in spectral peaks between the tumor cell subpopulations. The identity of the cellular components responsible for these spectral characteristics are unknown, but some clearly arise from differences in the extractable lipids present in the tumor cell subpopulations. Further study will be required to determine if the spectral differences described in this preliminary report are directly related to the known biochemical characteristics of the highly metastatic clone, and if the observations have general relevance to metastatic potential or are a singular feature of these cells. However, these initial results suggest that manipulation of factors which allow unmasking of spectral detail combined with the use of prescribed tumor cell subpopulations may aid in using proton NMR to identify and define biochemical or structural differences related to the metastatic potential of tumor cells.     

Chromosome and DNA analyses of rat 13762NF mammary adenocarcinoma cell lines and clones of different metastatic potentials

Chromosome morphologies revealed by Giemsa-banded karyotypes and chromosome numbers were compared between parental tumor-, lymph node- and lung metastasis-derived rat 13762NF mammary adenocarcinoma cell lines and clones having different spontaneous metastatic potentials. Although chromosome numbers in the cell lines and clones generally correlated with DNA content by flow cytometry, ploidy did not correlate with spontaneous metastatic potentials. Chromosone number and DNA content drifted during prolonged in vitro growth in each of the cell lines and clones. Common chromosome rearrangements were found, confirming a common origin for all the cell lines and clones, and the frequency and appearance of the individual marker chromosomes fluctuated during in vitro growth. Karyotypic analyses revealed that the markers coinciding with phenotypic drift in spontaneous metastatic potential and other biological properties of parental tumor-derived clones MTC and MTF7 and lung metastasis-derived clone MTLn3 involved chromosomes 3, 4, 5, 6, and 8. Clone MTC exhibited a shift in several markers and an increase in metastatic potential at passage T20, while clone MTF7 displayed a lesser spontaneous metastatic potential at high passage (T34) concomitant with an increase in the frequency of certain marker chromosomes. Lung metastasis-derived clone MTLn3 also exhibited a shift in some marker chromosomes, colonization preference and metastatic potential to lung and lymph nodes at high tissue culture passages. The changes in marker chromosomes during in vitro passage of clones MTC and MTLn3 suggested the presence of at least two cell subpopulations which could be responsible for the observed shift in spontaneous metastatic properties. Karyotypic features of the 13762NF cell lines and clones indicate that subtle cytogenetic changes, in contrast to gross chromosomal abnormalities, may be more important in determining metastatic phenotype.     

应用蛋白质组学分析结直肠癌促泌素和糖调节蛋白78蛋白变化及其意义

目的分析结直肠癌、配对正常黏膜组织差异表达蛋白质,寻找与结直肠癌发生、发展相关的分子标记物。方法固相pH梯度双向凝胶电泳分离结直肠癌患者配对癌及正常黏膜组织的总蛋白,液相色谱串联质谱鉴定差异表达蛋白质点。半定量逆转录聚合酶链反应（RT—PCR）、Western blot及免疫组织化学（EnVision法）方法检测促泌素（SCGN）及糖调节蛋白78（GRP78）两个差异表达蛋白质及组织定位,并检测54例神经内分泌肿瘤SCGN的表达。结果比较结直肠癌、配对正常黏膜组织蛋白质表达图谱,共获得5倍以上差异蛋白质点35个,癌组织中表达上调15个、下调20个。质谱分析鉴定14个蛋白质。RT-PCR及Western blot证实结直肠癌组织SCGN低表达,免疫组织化学染色发现正常结直肠黏膜神经内分泌细胞及98％（53／54）的神经内分泌肿瘤SCGN阳性。GRP78蛋白在结直肠癌组织中的表达明显高于正常黏膜组织,但在mRNA水平上癌组织和正常组织之间差异无统计学意义。结论双向凝胶电泳结合质谱分析是筛选肿瘤异常表达蛋白质的有效手段,所获得的35个差异表达候选蛋白质可能与结直肠癌的发生、发展有关。SCGN是一个候选神经内分泌标记物,GRP78蛋白表达增高可能涉及翻译后修饰。     

鼻咽癌转移相关的分泌蛋白质的筛选

目的：筛选鼻咽癌（nasopharyngeal carcinoma, NPC）转移相关的分泌蛋白质,为阐明NPC转移机制以及筛选NPC转移分子标志物提供实验依据。方法：应用二维凝胶电泳（two-dimensional electrophoresis,2-DE）技术分离一对来自同一亲本,具有不同转移潜能的NPC细胞系5-8F和6-10B细胞的分泌蛋白质,图像分析识别差异表达的蛋白质点,基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)和电喷雾-四极杆-串联质谱(ESI-Q-TOF MS/MS)对差异表达的蛋白质点进行鉴定。Western印迹法检测差异分泌蛋白质nm23-H1在两株细胞中的表达水平。结果：建立了5-8F和6-10B分泌蛋白质的2-DE图谱,质谱分析鉴定出14个非冗余的分泌蛋白质,其中Oncoprotein18等蛋白质在高转移潜能NPC细胞系5-8F中的表达水平高于无转移潜能的NPC细胞系,而nm23-H1等蛋白质在5-8F细胞系中的表达水平低于6-10B细胞系。Western印迹分析证实了nm23-H1在5-8F和6-10B细胞分泌蛋白质中的差异表达水平。结论：所鉴定的14个非冗余的差异分泌蛋白质为研究NPC转移机制以及筛选NPC转移分子标志物提供了实验依据。     

Rapid effects of EGF on cytoskeletal structures and adhesive properties of highly metastatic rat mammary adenocarcinoma cells

In the highly metastatic rat mammary adenocarcinoma cell clone MTLn3, EGF induced increased adhesion to fibronectin while in the human epidermoid carcinoma cell line A431 EGF induced diminished adhesive properties. Flattening of cells with extensive formation of fllopodia was observed in MTLn3 cells within 5 min of EGF addition, while in A431 cells EGF induced rounding up and only occasional formation of filopodia. Immunofluorescent analysis revealed extension of microtubutes (MT) into the filopodia and Western blot analysis demonstrated an EGF-induced 2- to 3-fold increase in the amount of assembled tubulin in MTLn3 but not in A431 cells. In MTLn3, but only marginally in A431 cells, EGF treatment resulted in phosphorylation of a 280 kD cytoskeleton-associated protein, which was rapid and dose-dependent. These results suggest differential signal transduction pathways of cytoskeleton-associated EGFRs in highly metastatic MTLn3 as compared with A431 cells.     

Medullary thyroid carcinoma with poor differentiation and atypical radiographic pattern of metastasis

A diagnosis of sporadic medullary thyroid carcinoma (MTC) is complicated. On first diagnosis it may present with distant metastasis. There has been inconsistency regarding metastatic MTC tissue expression of calcitonin, its tumor marker. Adding to the difficulty is the fact that the radiographic pattern of pulmonary metastasis from MTC may vary substantially among patients. Herein is reported the case of a 73-year-old man who presented with two ill-defined pulmonary opacities, clinically resembling primary lung carcinoma. MTC was diagnosed on histopathology of tissue obtained from a total thyroidectomy. The pulmonary biopsy specimens were confirmed to be MTC metastasis on positive immunohistochemical staining of chromogranin-A and synaptophysin, even though only a few cells were stained for calcitonin. To the authors' knowledge this is the first reported case of MTC presenting initially as complex pulmonary metastasis with weakened expression of calcitonin in the metastatic lesion.     

14-3-3sigma negatively regulates the cell cycle, and its down-regulation is associated with poor outcome in intrahepatic cholangiocarcinoma

The 14-3-3sigma gene has been implicated in G2/M cell cycle arrest by p53, and the loss of 14-3-3sigma protein expression has been reported in diverse human cancers. However, the role of 14-3-3sigma in the signaling pathway of the cell cycle in the progression of intrahepatic cholangiocarcinoma has not been well understood. To clarify the role of 14-3-3sigma, we examined the protein expressions of 14-3-3sigma, cyclin B1, and p53 in 93 cases of intrahepatic cholangiocarcinoma by immunohistochemical staining. We also examined the correlation between these expressions and survival rate and clinicopathologic factors such as sex, age, tumor grade (ie, pathologic differentiation, tumor size, lymphatic permeation, vascular invasion, perineural invasion, lymph node metastasis), and tumor stage. Positive 14-3-3sigma protein expression (>30% of tumor cells) was observed in 67.7% (63/93) of cases of intrahepatic cholangiocarcinoma and was inversely correlated with cyclin B1 expression. No correlation was found between 14-3-3sigma expression and p53 expression or clinicopathologic factors; however, decreased 14-3-3sigma expression was an independent prognostic factor by multivariate survival analysis (P = .0282). Extensive methylation of 14-3-3sigma was found by methylation-specific polymerase chain reaction and sequence; however, no significant correlation was detected between methylation states and protein expression. These results indicate that depressed 14-3-3sigma protein is involved in the uncontrolled cell cycle in intrahepatic cholangiocarcinoma and that the decreased expression of 14-3-3sigma protein is a significant indicator of poor prognosis for patients with intrahepatic cholangiocarcinoma.     

EGF stimulates lamellipod extension in metastatic mammary adenocarcinoma cells by an actin-dependent mechanism

Changes in lamellipod extension and chemotaxis in response to EGF were analysed for MTLn3 cells (a metastatic cell line derived from the 13762NF rat mammary adenocarcinoma). Addition of EGF produced a cessation of ruffling followed by extension of hyaline lamellipods containing increased amounts of F-actin at the growing edge. A non-metastatic cell line (MTC) derived from the same tumor did not show such responses. Lamellipod extension was maximal within 5 min, followed by retraction and resumption of ruffling. Maximal area increases due to lamellipod extension occurred at about 5 nM EGF. Chemotactic and chemokinetic responses, measured using a microchemotaxis chamber, were also greatest at 5 nm. Cytochalasin D inhibited EGF-stimulated responses including lamellipod extension, increases in F-actin in lamellipods, and chemotaxis. Nocodazole affected chemotaxis at higher concentrations but not EGF-induced lamellipod extension. We conclude that polymerization of F-actin at the leading edges of lamellipods is necessary for extension of lamellipods and chemotaxis of MTLn3 cells in response to EGF. The motility and chemotaxis responses of this metastatic cell line have strong similarities to those seen in well-characterized chemotactic cells such as Dictyostelium and neutrophils.     

An improved method to study NK-independent mechanisms of MTLn3 breast cancer lung metastasis

To study the tumor cell autonomous processes of metastasis, an in vivo tumor metastasis model is required that excludes the involvement of the innate immune system. For this purpose we used the established syngeneic MTLn3 cell - Fischer 344 tumor model. MTLn3 cells are efficiently eradicated by NK cells in vivo. Using isolated cell systems, we provide evidence for apoptosis-induction by IL-2 activated NK cells, but not T-cells, despite the expression of MHC class I. This is largely mediated by the perforin/granzyme B pathway in MTLn3 cells in a caspase-dependent manner. Temporal in vivo depletion of NK cells by an antibody-based method, dramatically improved colonization of the lungs by MTLn3 cells, from 5 metastases in the untreated animals to 130 metastases in the NK-depleted animals. Thus, we improved the syngeneic MTLn3-Fischer 344 tumor model by temporal depletion of NK cells of which the advantages over the use of immunodeficient animals are evident.     

An improved model to study tumor cell autonomous metastasis programs using MTLn3 cells and the Rag2<Superscript>−/−</Superscript> γc<Superscript>−/−</Superscript> mouse

The occurrence of metastases is a critical determinant of the prognosis for breast cancer patients. Effective treatment of breast cancer metastases is hampered by a poor understanding of the mechanisms involved in the formation of these secondary tumor deposits. To study the processes of metastasis, valid in vivo tumor metastasis models are required. Here, we show that increased expression of the EGF receptor in the MTLn3 rat mammary tumor cell-line is essential for efficient lung metastasis formation in the Rag mouse model. EGFR expression resulted in delayed orthotopic tumor growth but at the same time strongly enhanced intravasation and lung metastasis. Previously, we demonstrated the critical role of NK cells in a lung metastasis model using MTLn3 cells in syngenic F344 rats. However, this model is incompatible with human EGFR. Using the highly metastatic EGFR-overexpressing MTLn3 cell-line, we report that only Rag2^−/−γc^−/− mice, which lack NK cells, allow efficient lung metastasis from primary tumors in the mammary gland. In contrast, in nude and SCID mice, the remaining innate immune cells reduce MTLn3 lung metastasis formation. Furthermore, we confirm this finding with the orthotopic transplantation of the 4T1 mouse mammary tumor cell-line. Thus, we have established an improved in vivo model using a Rag2^−/− γc^−/− mouse strain together with MTLn3 cells that have increased levels of the EGF receptor, which enables us to study EGFR-dependent tumor cell autonomous mechanisms underlying lung metastasis formation. This improved model can be used for drug target validation and development of new therapeutic strategies against breast cancer metastasis formation.     

Ezrin in primary cutaneous melanoma.

Ezrin is a member of the ezrin-radixin-moesin family of proteins that link the actin-containing cytoskeleton to the plasma membrane. Ezrin is also connected to signaling molecules involved in the regulation of cell survival, proliferation and migration. Here, we examined the expression of ezrin in 95 primary cutaneous melanomas and correlated ezrin expression with conventional prognostic factors and biomarkers. From 12 patients metastatic tissue samples were also examined. In addition to ezrin staining, Mib-1 proliferation antigen, p53 and Bcl-2 were evaluated. Ezrin immunoreactivity was seen in most tumors; only 19 (20%) melanomas were negative. A total of 48 (51%) tumors had weak immunoreactivity and 28 (29%) strong immunoreactivity. The intensity of ezrin immunoreactivity was associated with tumor thickness (Breslow, P=0.0008) and with tumor invasion level (Clark, P=0.004), thicker tumors having stronger immunoreactivity. Also, there was a correlation between higher Mib-1 index in tumors and strong ezrin expression. All metastatic samples (n=12) showed positive ezrin immunoreactivity. In univariate analysis of survival, patients (n=76) with positive ezrin immunoreactivity had worse clinical disease behavior than those (n=19) without ezrin immunoreactivity, but the difference was not significant (P=0.19). In multivariate analysis of survival, the ezrin immunoreactivity was not a significant marker. The results indicate that ezrin is expressed in most primary melanomas of the skin and in all metastatic tumors. Ezrin expression correlates with tumor thickness and level of invasion suggesting an association between ezrin expression and tumor progression.     

人肿瘤转移抑制基因TMSG-1单克隆抗体的制备、鉴定及在肿瘤检测中的应用

目的　明确人肿瘤转移抑制基因TMSG- 1蛋白分子量大小和细胞定位,制备和鉴定TMSG- 1单克隆抗体,并探讨其在临床肿瘤标本检测的价值。方法　采用多肽固相合成法 (Fmoc法 )合成 15肽TMSG -1抗原片段,并与匙孔槭血蓝蛋白 (mcKLH)偶联,免疫BALB/C小鼠,采用细胞融合、ELISA法检测和快速有限稀释法筛选和制备单克隆抗体,并应用Western印迹和免疫组织化学ABC法染色进行抗体鉴定和肿瘤检测。结果　筛选出抗TMSG- 1抗体的单克隆杂交瘤细胞系C8,免疫球蛋白亚类测定为IgM。半抗原竞争抑制实验证实该抗体是特异的抗TMSG- 1抗体。Western印迹显示不转移癌细胞亚系 2B4和LH7有明显的相对分子质量为 45 .000蛋白条带,而高转移亚系 1E8和BE1则条带很弱。细胞免疫组织化学染色显示 2B4和LH7强阳性,为细胞膜和细胞质着色;而 1E8和BE1则为阴性。石蜡切片ABC法检测 52例乳腺癌和 41例结肠癌组织中TMSG- 1蛋白表达发现:未转移的癌组织多为中度阳性或强阳性,中度以上阳性率分别为 36% (乳腺癌 )和 52. 4% (结肠癌 );而转移的癌组织多为弱阳性或阴性, 中度以上阳性率分别为 7 .4% (乳腺癌 )和 35% (结肠癌 ),统计学分析表明TMSG 1在未转移癌组织中的表达显著高于转移性癌组织 (P<0 .05)。结论　成功制备了抗人肿瘤转移抑制基     

miRNA‐214 is related to invasiveness of human non‐small cell lung cancer and directly regulates alpha protein kinase 2 expression

The prognosis of non‐small cell lung cancer (NSCLC) is poor, since it has often metastasized to distant organs by the time of diagnosis. Therefore, biomarkers predicting metastasis are crucial. miRNAs play important roles in the regulation of different tumor cell processes, including metastasis. We recently showed that miRNA‐214 is linked to a radioresistant phenotype of NSCLC. miRNA‐214 has been linked to metastasis in other tumor types. Therefore, we examined the role of miRNA‐214 in the metastatic potential of NSCLC. We showed that downregulation of miRNA‐214 increased invasive potential, and conversely, overexpression of miRNA‐214 decreased invasiveness of NSCLC cells in vitro. Gene expression and bioinformatic analyses of NSCLC cells with ablated miRNA‐214, identified a number of metastasis‐related target genes, including pregnancy‐associated plasma protein A (PAPP‐A), alpha protein kinase 2 (ALPK2), cyclin‐dependent kinase 6 (CDK6) and tumor necrosis‐factor alpha‐induced protein 3 (TNFAIP3). These were validated on mRNA and protein level to be regulated by miRNA‐214. Through immunoprecipitation we showed that only ALPK2 is directly regulated by miRNA‐214. We also examined the protein expression of these four genes in NSCLC tumors with respect to metastatic potential. These results showed that NSCLC tumors express these proteins at moderate‐high levels in the nucleus, cytoplasm and/or plasma membrane although with no significant correlation to the overall survival or the metastatic potential of the patients. However, we also showed that the membrane‐localized PAPP‐A had a higher expression level compared to the cytoplasm‐localized. In conclusion, we show that low miRNA‐214 expression is linked to a higher invasive potential of NSCLC cells. © 2013 Wiley Periodicals, Inc.     

G-protein coupled receptor-associated sorting protein 1 (GASP-1), a potential biomarker in breast cancer

An innovative “2-D high performance liquid electrophoresis” (2-D HPLE) technology was used to identify serum biomarkers associated with the early stage of breast cancer in addition to other more advanced stages. 2-D HPLE is a newly developed electrophoretic technology that separates 100s of serum albumin complexes on a polyvinyl membrane based on their surface charges. Association of cancer proteins or their fragments (biomarkers) with pre-existing albumin complexes in the blood of cancer patients results in altered mobility on the membrane. Using 2-D HPLE we identified that a specific fragment of G-protein coupled receptor-associated sorting protein 1 (GASP-1) was present in the sera of patients with early stage disease but absent in sera of normal patients. GASP-1 has been shown to modulate lysosomal sorting and functional down-regulation of a variety of G-protein coupled receptors in neuronal cells. However, no reports have linked GASP-1 to breast cancer pathogenesis. GASP-1 was detected in tumor extracts of 7 cases of Stage 2 and Stage 3 breast cancers, but not in adjacent normal tissue as revealed by western blot analysis using an antibody developed against a GASP-1 peptide identified by our 2-D HPLE technology. Using this antibody, we immunohistochemically detected over-expression of GASP-1 in all of 107 cases of archived ductal breast carcinoma tumor samples, while normal adjacent breast tissue from 12 cases of ductal carcinoma showed little or no staining. Additionally, all 10 cases of metastatic breast carcinoma present in lymph nodes were positive. Strong positive GASP-1 staining was observed in all tumor tissue including ductal carcinoma in situ (DCIS) and invasive ductal carcinoma. Additionally, we observed a wide spectrum of enhanced staining of premalignant ductal epithelial cells present in benign ducts and those found in atypical ductal hyperplasia (ADH). These studies identify GASP-1 as a potential new serum and tumor biomarker for breast cancer and suggest that GASP-1 may be a novel target for the development of breast cancer therapeutics.     

Immunohistochemical analysis of 14-3-3 sigma and related proteins in hyperplastic and neoplastic breast lesions, with particular reference to early carcinogenesis

In order to confirm the role of 14-3-3 sigma (sigma) as a tumor suppressor in breast carcinogenesis, we have studied the expression of 14-3-3sigma immunohistochemically in usual ductal hyperplasia (UDH), ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) breast lesions. Immunostaining for estrogen receptor alpha (ERalpha), p53 and estrogen-responsive RING finger protein (Efp) was also carried out. Immunohistochemically, expression of 14-3-3sigma was seen in 92% UDH lesions and gradually decreased from 65% in DCIS to 23% in IDC. The expression of ERalpha decreased gradually from UDH to DCIS to IDC, while p53 showed an inverse staining pattern to that of ERalpha. The expression of Efp showed no significant difference among the three breast lesions. Hence, the present immunohistochemical study confirmed 14-3-3sigma as a tumor suppressor in breast carcinogenesis. A similar immunohistochemical analysis was then carried out on columnar cell hyperplasia with atypia (CCHA), in which the expression pattern of tumor suppressor 14-3-3sigma, ERalpha and p53 suggested that it might be possible that CCHA is a precancerous lesion.     

Expression of reactive oxygen species-related proteins in metastatic breast cancer is dependent on the metastatic site

This study was performed to investigate the expression of reactive oxygen species (ROS)-related proteins and to analyze the implications for primary and metastatic breast cancer. We constructed a tissue microarray containing 143 metastatic breast cancers (52 lung metastases, 38 bone metastases, 37 brain metastases, and 16 liver metastases) and performed immunohistochemical staining for ROS-related proteins (catalase, GSTπ, TxNIP, and MnSOD). Analysis of ROS-related protein expression in metastatic breast cancers according to the metastatic sites revealed site-specific expression patterns. The expression of tumoral catalase was lower in bone metastases (P = 0.012), and stromal GSTπ expression was higher in bone and liver metastases (P < 0.001). The highest ROS activation status was observed for lung metastases, while non-activated ROS was observed for bone metastases (P = 0.001). Primary cancers were positive for stromal GSTπ, but a subset of lung metastases were negative (P = 0.021). Univariate analysis revealed that shorter overall survival (OS) was associated with negative catalase expression of the tumor (P = 0.026). Furthermore, univariate analyses according to the metastatic sites revealed that shorter OS was associated with TxNIP-positive tumors (P = 0.032) and the expression of stromal catalase (P = 0.032) in brain metastases. Tumors that were negative for MnSOD expression (P < 0.001) but positive for stromal catalase expression (P = 0.022) were associated with shorter OS in patients with liver metastases. In conclusion, cancer cells and stromal tissues showed different ROS-related protein expression patterns according to the metastatic site. In addition, the expression of ROS-related proteins is associated with patient prognosis.