Correction to: Identification of the functional role of peroxiredoxin 6 in the progression of breast cancer.

After the publication of this work [1] an error in Fig. 1c was brought to our attention: the Western blots for PRDX6 and ß-actin were similar to those shown in lanes 5-6 of Fig. 4g. To verify these findings, we have repeated this experiment and the results are shown in a new Fig. 1c below. The repeated experimental results are consistent with the previously reported findings in the original study [1] and the functional role for PRDX6 in malignant progression of human cancer including breast cancer has been widely documented and recognized in numerous other studies [2]. We apologize for the error. However, this correction does not affect the conclusions of the article.

Extracellular 5'-nucleotidase (CD73) promotes human breast cancer cells growth through AKT/GSK-3ß/ß-catenin/cyclinD1 signaling pathway.

To identify the role and to explore the mechanism of extracellular 5'-nucleotidase (CD73) in human breast cancer growth, CD73 expression was measured firstly in breast cancer tissues and cell lines, and then interfered with or over-expressed by recombinant lentivirus in cell lines. Impacts of CD73 on breast cancer cell proliferation and cell cycle were investigated with colony formation assay, CCK-8 and flow cytometry. The relationship between CD73 and AKT/GSK-3ß/ß-catenin pathway was assessed with adenosine, adenosine 2A receptor antagonist (SCH-58261), adenosine 2A receptor agonist (NECA), CD73 enzyme inhibitor (APCP) and Akt inhibitor (MK-2206). Moreover, the effect of CD73 on breast cancer growth in vivo was examined with human breast cancer transplanting model of nude mice. The results showed that the expression of CD73 was high in breast cancer tissues and increased with advanced tumor grades and lympho-node status. CD73 expression was higher in more malignant cells, and CD73 overexpression promoted breast cancer cell proliferation in both in vivo and in vitro. It activated AKT/GSK-3ß/ß-catenin/cyclinD1 signaling pathway through CD73 enzyme activity and other mechanism.

Suberoyl bis-hydroxamic acid enhances cytotoxicity induced by proteasome inhibitors in breast cancer cells.

BACKGROUND: Suberoyl bis-hydroxamic acid (SBHA) is a histone deacetylase (HDAC) inhibitor and exerts anti-growth effects in several malignancies including breast cancer. Proteasome inhibitors such as Bortezomib and MG-132 constitute novel anticancer agents. In this study, we investigated the synergistic antitumour activity of SBHA in combination with proteasome inhibitors. METHODS: MCF-7 and MDA-MB-231 breast cancer cells were treated with SBHA, Bortezomib, and MG-132 alone or in combination for 72 h. Cell proliferation, colony formation, apoptosis and gene expression changes were examined. RESULTS: SBHA, Bortezomib, and MG-132 alone significantly inhibited the proliferation and colony formation and induced apoptosis in MCF-7 and MDA-MB-231 cells. Combined treatment showed a good synergistic antitumour effect against breast cancer cells. The p53 protein level was significantly elevated by combined treatment with SBHA and proteasome inhibitors. Moreover, combined treatment increased the expression of Bax, Bcl-xS, and Bak and decreased the expression of Bcl-2. Combination of SBHA with proteasome inhibitors causes synergistic anticancer effects on breast cancer cells. The potential molecular mechanism may involve induction of p53 and modulation of the Bcl-2 family proteins. CONCLUSION: These findings warrant further investigation of the therapeutic benefits of combination of SBHA with proteasome inhibitors in breast cancer.

COX-2 promotes breast cancer cell radioresistance via p38/MAPK-mediated cellular anti-apoptosis and invasiveness.

Radioresistance is one of the major barriers to improve the survival rate of breast cancer patients. Cyclooxygenase 2 (COX-2) is usually overexpressed in highly invasive and metastatic breast cancer, which may indicate an association with breast cancer radioresistance. The function role of COX-2 was investigated by using a radioresistant breast cancer cell line MDA-MB-231/RR10 and its parental cell line MDA-MB-231 cells before or after COX-2 was silenced by a specific small hairpin RNA (shRNA). The cell proliferation, migration, invasion, colony formation, and apoptosis were measured by CCK-8, scratch-wound, transwell, clone formation assay, and flow cytometry. Protein and mRNA expression were analyzed by Western blot and quantitative reverse transcriptase-polymerase chain reaction. COX-2 is upregulated in MDA-MB-231/RR10 cells compared with in MDA-MB-231 cells, and silencing of COX-2 expression by shRNA in MDA-MB-231/RR10 cells decreases the expression of Bcl-2 and Bcl-XL, but increases the proapoptotic protein BAK, leading to the increased apoptosis following treatment with γ-irradiation in comparison with those in control cells. Silencing of COX-2 also increases the expression of ß-catenin and E-cadherin, two anti-invasion proteins, resulting in reduced cell migration and invasion tested by transwell chambers and wound-healing assays. Further study demonstrated that COX-2-induced radioresistance is negatively regulated through the phosphorylation of p38 at Tyr182, and that the phosphorylation of p38 induced by TNF-alpha reduces the expression of Bcl-2, BCL-XL, but increases ß-catenin and E-cadherin, leading to the decreased invasiveness of cells. Our data suggest that COX-2, p38, Bcl-2, Bcl-XL, ß-catenin, and E-cadherin may be considered as potential therapeutic targets against radioresistant breast cancer.

FOXC1, a target of polycomb, inhibits metastasis of breast cancer cells.

Polycomb group (PcG) proteins have recently been shown related to cancer development. The PcG protein EZH2 is involved in progression of prostate and breast cancers, and has been identified as a molecular marker in breast cancer. Nevertheless, the molecular mechanism by which PcG proteins regulate cancer progression and malignant metastasis is still unclear. PcG proteins methylate H3K27 in undifferentiated epithelial cells, resulting in the repression of differentiation genes such as HOX. FOXC1 is a member of the Forkhead box transcription factor family, which plays an important role in differentiation, and is involved in eye development. We discovered in this study that the expression of FOXC1 gene was negatively correlated to that of PcG genes, i.e., Bmi1, EZH2, and SUZ12, in MCF-7 and MDA-MB-231 cells. To investigate the regulatory effects of PcG proteins on FOXC1 gene, the two cell lines were transfected with either expression plasmids or siRNA plasmids of Bmi1, EZH2, and SUZ12, and we found that PcGs, especially EZH2, could repress the transcription of FOXC1 gene. Chromatin immunoprecipitation (ChIP) assay showed that histone methylation and acetylation modifications played critical roles in this regulatory process. When FOXC1 was stably transfected into MDA-MB-231 cells, the migration and invasion of the cells were repressed. Moreover, the tumorigenicity and the spontaneous metastatic capability regulated by FOXC1 were determined by using an orthotropic xenograft tumor model of athymic mice with the FOXC1-MDA-MB-231HM and the GFP-MDA-MB-231HM cells, and the results showed that FOXC1 in MDA-MB-231HM cells inhibited migration and invasion in vitro and reduced the pulmonary metastasis in vivo. Data presented in this report contribute to the understanding of the mechanisms by which EZH2 participates in tumor development.

Expression of CXCL14 and its anticancer role in breast cancer.

CXCL14, also known as breast and kidney-expressed chemokine, was initially identified as a chemokine highly expressed in the kidney and breast. The exact function of CXCL14 in human breast cancer is still unclear, although it has been testified to play an anti-tumor role in other tumors, including head and neck squamous cell carcinoma, lung cancer, prostate cancer, and so on. In this study, we tried to demonstrate the relationship between CXCL14 and breast cancer. CXCL14 expressions were detected by reverse transcription-PCR and western blot in 2 normal breast epithelial cell lines and 6 breast cancer cell lines. The effects of CXCL14 on the proliferation and invasion in vitro were tested using the CXCL14-overexpressing cells (MDA-MB-231HM-CXCL14) which were established by stable transfection. We established an orthotropic xenograft tumor model in SCID mice using the MDA-MB-231HM-CXCL14 cells and explored the influence of CXCL14 overexpression on tumor growth and metastasis in vivo. Furthermore, we detected the protein level of CXCL14 in 208 breast cancer patients by immunohistochemistry and discussed the correlation between CXCL14 and the prognosis of breast cancer. CXCL14 mRNA expression is lower in breast cancer cell lines, and MDA-MB-231HM express the lowest levels of CXCL14 mRNA. Overexpression of CXCL14 inhibited cell proliferation and invasion in vitro and attenuated xenograft tumor growth and lung metastasis in vivo. CXCL14 protein level is positively correlated to the overall survival of all patients as well as the patients with lymph node metastasis, and it has a negative correlation with the lymph node metastasis. Our study showed for the first time that CXCL14 is a negative regulator of growth and metastasis in breast cancer. The re-expression or up-regulation of this gene may provide a novel strategy in breast cancer therapy in the future.

The chemokine receptor CCR4 promotes tumor growth and lung metastasis in breast cancer.

Increasing evidence has shown that chemokines and chemokine receptors are associated with tumor growth and metastasis. CCR4, an important chemokine receptor for regulating immune homeostasis, is thought to be involved in hematologic malignancies and has also recently implicated in some solid tumors, such as gastric cancer. The possible role of CCR4 in breast cancer has not been well elucidated. In this study, we show that CCR4 is differentially expressed in human breast cancer cell lines. Specifically, we find that CCR4 is overexpressed in breast cancer cell lines with high metastatic potential. More importantly, we used a combination of overexpression and RNA interference to demonstrate that CCR4 promotes breast tumor growth and lung metastasis in mice. Furthermore, we find that microvessel density is significantly increased in tumors formed by CCR4-overexpressing cells and decreased in those formed by CCR4-knockdown cells. We find that overexpression of CCR4 can enhance the chemotactic response of breast cancer cells to CCL17. However, the expression of CCR4 does not affect the proliferation of breast cancer cells in vitro. Furthermore, we show that CCR4 expression is positively correlated with HER2 expression, tumor recurrence and lymph node, lung and bone metastasis (P < 0.05). Multivariate analysis showed that CCR4 expression is a significant independent prognostic factor for overall survival (P = 0.036) but not for disease-free survival in patients with breast cancer (P = 0.071). Survival analysis indicated a strong association between CCR4 expression and lower overall survival (P = 0.0001) and disease-free survival (P = 0.016) in breast cancer.

Correlation between Duffy blood group phenotype and breast cancer incidence.

BACKGROUND: Different ethnicities have different distribution of Duffy blood group (DBG) phenotypes and different breast cancer morbidity. A study in our lab demonstrated that Duffy antigen/receptor for chemokines (DARC, also known as DBGP, the Duffy protein phenotype), led to the inhibition of tumorigenesis. Therefore, we tested the hypothesis that DBGP is correlated with breast cancer occurrence. METHODS: DBGP proteins were examined by indirect antiglobulin testing with anti-FYa and anti-FYb antibodies. The phenotypes were classified into four groups according to the agglutination reactions: FYa + FYb+, FYa + FYb-, FYa-FYb + and FYa-FYb-. The phenotypes and pathological diagnosis of consecutively hospitalized female patients (n = 5,022) suffering from breast cancer at the Shanghai Cancer Hospital and Henan Province Cancer Hospital were investigated. The relationships between DBGP expression with breast cancer occurrence, axillary lymph status, histological subtype, tumor size pathological grade and overall survival were analyzed. RESULTS: The incidence of breast cancer was significantly different between FYa + FYb + (29.8%), FYa + FYb- (33.2%), FYa-FYb + (45.6%) and FYa-FYb- (59.1%; P = 0.001). Significant different numbers of breast cancer patients had metastases to the axillary lymph nodes in the FYa + FYb + group (25.1%), FYa + FYb- (36.9%), FYa-FYb + (41.0%) and FYa-FYb- (50.0%, (P = 0.005). There was a statistical significance (p = 0.022) of the overall survival difference between patients with difference phenotypes. No significant difference was observed in cancer size (t-test, p > 0.05), histological cancer type (Fisher's exact test, p > 0.05) or histological grade (Fisher's exact test, p > 0.05) between every each DBGP group. CONCLUSIONS: DBGP is correlated with breast cancer incidence and axillary lymph node metastasis and overall survival. Further investigations are required to determine the underlying mechanism of Duffy blood group phenotype on breast cancer risk.

Coexpression of atypical chemokine binders (ACBs) in breast cancer predicts better outcomes.

Some evidence suggests that atypical chemokine binders (ACBs) including DARC, D6, and CCX-CKR play an important role in inhibiting invasion and metastasis of cancer cells; however, their expression in breast cancer has not been well characterized. The purpose of this study was to determine the predictive value of ACBs for relapse-free survival and overall survival in breast cancer. The expressions of the three molecules were analyzed immunohistochemically in a total of 558 consecutive breast specimens comprising 12 normal breast tissues, 29 noninvasive (carcinoma in situ), and 517 invasive breast carcinoma and their relationships to clinicopathological features and survival were investigated in invasive breast cancer. Coexpression of ACBs in invasive breast carcinoma (55.9%) was much lower that of noninvasive breast carcinoma (93.1%) and normal breast tissue (100.0%), P = 0.0004, 0.0096, respectively. Their separate stainings in invasive cancer were significantly conversely correlated with lymph node status and tumor stage. In univariate analysis, the three proteins and their coexpression were significantly associated with higher relapse-free survival and overall survival. In multivariate analysis, each of these molecules was favorable for relapse-free survival, but not overall survival. Surprisingly, their coexpression was not only independently prognostic factor for relapse-free survival (RR = 0.182, 95% CI: 0.101-0.327, P < 0.001), but also for overall survival (RR = 0.271, 95% CI: 0.081-0.910, P = 0.035). These findings highlight that the multiple loss of ACBs may occur during the development of tumorigenesis and their coexpression in breast cancer is predictive of favorable outcomes.

Involvement of a novel chemokine decoy receptor CCX-CKR in breast cancer growth, metastasis and patient survival.

PURPOSE: The biological axes of chemokines and chemokine receptors, such as CXCR4/CXCL12, CCR7/CCL19 (CCL21), CCR9/CCL25, and CXCR5/CXCL13, are involved in cancer growth and metastasis. This study is aimed at the potential regulatory role of atypical chemokine binder CCX-CKR, as a scavenger of CCL19, CCL21, CCL25, and CXCL13, in human breast cancer. EXPERIMENTAL DESIGN: The role of CCX-CKR in human breast cancer was investigated in cell lines, animal models, and clinical samples. RESULTS: Overexpression of CCX-CKR inhibited cancer cell proliferation and invasion in vitro and attenuated xenograft tumor growth and lung metastasis in vivo. CCX-CKR can be regulated by cytokines such as interleukin-1beta, tumor necrosis factor-alpha, and IFN-gamma. Lack or low expression of CCX-CKR correlated with a poor survival rate in the breast cancer patients. A significant correlation between CCX-CKR and lymph node metastasis was observed in human breast cancer tissues. CCX-CKR status was an independent prognostic factor for disease-free survival in breast cancer patients. CONCLUSION: We showed for the first time that CCX-CKR is a negative regulator of growth and metastasis in breast cancer mainly by sequestration of homeostatic chemokines and subsequent inhibition of intratumoral neovascularity. This finding may lead to a new therapeutic strategy against breast cancer.

Chemokine decoy receptor d6 plays a negative role in human breast cancer.

Chemokine binding protein D6 is a promiscuous decoy receptor that can inhibit inflammation in vivo; however, the role it plays in cancer is not well known yet. In this study, we showed for the first time that human breast cancer differentially expressed D6 and the expression could be regulated by some cytokines. More importantly, overexpression of D6 in human breast cancer cells inhibits proliferation and invasion in vitro and tumorigenesis and lung metastasis in vivo. This inhibition is associated with decreased chemokines (e.g., CCL2 and CCL5), vessel density, and tumor-associated macrophage infiltration. Furthermore, D6 expression is inversely correlated to lymph node metastasis as well as clinical stages, but positively correlated to disease-free survival rate in cancer patients. Therefore, D6 plays a negative role in the growth and metastasis of breast cancer.

CXCR2 promotes breast cancer metastasis and chemoresistance via suppression of AKT1 and activation of COX2.

Metastasis and chemoresistance are two major causes of breast cancer death. We show here that the chemokine receptor CXCR2 was overexpressed in breast cancer cell lines and tissues. CXCR2 promoted anti-apoptosis, anti-senescence, and epithelial-to-mesenchymal transition (EMT) of breast cancer cells, leading to the enhanced metastasis and chemoresistance. Further study suggested that AKT1 and cyclooxygenase-2 (COX2; PTGS2) might mediate the CXCR2 signaling to inversely control the breast cancer metastasis and chemoresistance through the regulation of EMT, apoptosis, and senescence. Analyses of clinical data indicate that the high expression of CXCR2 was correlated with the high expression of COX2 and the low expression of AKT1, P85α, E-cadherin, and ß-catenin in cancer tissues. Poor outcomes were associated with the high expression of CXCR2 or COX2 while favorable survivals were associated with the high expression of P85α, AKT1, or E-cadherin in all cancer patients. Cox multivariate analysis demonstrated that CXCR2, COX2, and AKT1 could be independent predictors for disease free survivals. All these data suggest that CXCR2 promotes breast cancer metastasis and chemoresistance via suppressing AKT1 and activating COX2. Thus, antagonists of the CXCR2 signaling molecules may be used to treat breast cancer patients particularly with high metastasis and chemoresistance.

Identification of the functional role of peroxiredoxin 6 in the progression of breast cancer.

INTRODUCTION: The molecular mechanisms involved in breast cancer metastasis still remain unclear to date. In our previous study, differential expression of peroxiredoxin 6 was found between the highly metastatic MDA-MB-435HM cells and their parental counterparts, MDA-MB-435 cells. In this study, we investigated the effects of peroxiredoxin 6 on the proliferation and metastatic potential of human breast cancer cells and their potential mechanism. METHODS: Expression of peroxiredoxin 6 in the highly metastatic MDA-MB-231HM cells was investigated by RT-PCR, real-time PCR and western blot. A recombinant expression plasmid of the human peroxiredoxin 6 gene was constructed and transfected into MDA-MB-231 and MDA-MB-435 cells. The effects of peroxiredoxin 6 on the proliferation and invasion of MDA-MB-231 and MDA-MB-435 cells were investigated by the Cell Counting Kit-8 method, colony-formation assay, adhesion assay, flow cytometry and invasion assay in vitro. miRNA was used to downregulate the expression of peroxiredoxin 6. Genes related to the invasion and metastasis of cancer were determined by RT-PCR, real-time PCR and western blot. The tumorigenicity and spontaneously metastatic capability regulated by peroxiredoxin 6 were determined using an orthotopic xenograft tumor model in athymic mice. RESULTS: Overexpression of peroxiredoxin 6 in MDA-MB-231HM cells compared with their parental counterparts was confirmed. Upregulation of peroxiredoxin 6 enhanced the in vitro proliferation and invasion of breast cancer cells. The enhancement was associated with decreasing levels of tissue inhibitor of matrix metalloproteinase (TIMP)-2 and increasing levels of the urokinase-type plasminogen activator receptor (uPAR), Ets-1 (E26 transformation-specific-1), matrix metalloproteinase (MMP)-9 and RhoC (ras homolog gene family, member C) expression. The results were further demonstrated by RNA interference experiments in vitro. In an in vivo study, we also demonstrated that peroxiredoxin 6-transfected breast cancer cells grew much faster and had more pulmonary metastases than control cells. By contrast, peroxiredoxin 6 knockdown breast cancer cells grew more slowly and had fewer pulmonary metastases. Effects similar to those of peroxiredoxin 6 on the uPAR, Ets-1, MMP-9, RhoC and TIMP-2 expression observed in in vitro studies were found in the in vivo study. CONCLUSION: Overexpression of peroxiredoxin 6 leads to a more invasive phenotype and metastatic potential in human breast cancer, at least in part, through regulation of the levels of uPAR, Ets-1, MMP-9, RhoC and TIMP-2 expression.

Overexpression of Ecto-5'-nucleotidase (CD73) promotes T-47D human breast cancer cells invasion and adhesion to extracellular matrix.

Ecto-5'-nucleotidase (CD73) was overexpressed in malignancies of epithelial origin and was involved in a variety of cellular processes such as cytoprotection and anti-inflammation. In the present study, human mammary T-47D cells were transfected with pcDNA-NT5E to establish a CD73 overexpressed cell model. Short small interfering RNA (siRNA) was used to silence the epidermal growth factor receptor (EGFR) gene. Real-time PCR, RT-PCR and western-blot were used to study CD73 and EGFR expression. Surface CD73 activity was assessed by quantifying the conversion of etheno-AMP to ethenoadenosine via HPLC. Interleukin (IL)-8 mRNA and protein expression were analyzed by RT-PCR and ELISA. Cell motility, invasiveness and adhesion to extracellular matrix (ECM) were measured by in vitro invasion and adhesion assay before and after transfection. We demonstrated that abilities of migration, invasions and adhesion to ECM in pcDNA-NT5E transfected T-47D cells increased significantly, which can be blocked by CD73 inhibitor alpha, beta-methylene ADP(APCP). Addition of adenosine reversed the effects of APCP. The mRNA and protein expression of EGFR and IL-8 increased in pcDNA-NT5E transfected T-47D cells. EGFR siRNA down-regulated EGFR expression dramatically, leading to migration and invasion activities inhibition in pcDNA-NT5E transfected T-47D cells. Our results suggest that up-regulated adenosine production, EGFR and IL-8 expression due to overexpressed CD73 may involved in CD73-promoted breast cancer metastasis.

RNA interference of ecto-5'-nucleotidase (CD73) inhibits human breast cancer cell growth and invasion.

Metastasis is a leading cause of mortality and morbidity in breast cancer. Recently, dramatic overexpression of ecto-5'-nucleotidase (CD73), a glycosylphosphatidylinositol-anchored cell surface protein has been found in estrogen receptor-negative [ER (-)] breast cancer cell lines and in clinical samples. In this study, CD73 small interfering RNA (siRNA) plasmid was constructed and stably transfected into breast cancer cell MB-MDA-231 to determine the role of CD73 in breast cancer metastasis and the possible mechanism. Our study demonstrates that CD73 siRNA effectively inhibits CD73 gene expression at mRNA and protein level in MB-MDA-231 cells, leading to in vivo and in vitro growth suppression, prevention of adhesion to extracellular matrix (ECM), and inhibition of invasion and migration. These properties correlate with inhibition of matrix metalloproteinase (MMP)-2 and MMP-9 expression and activity as well as reduction of epidermal growth factor receptor (EGFR) expression. Demonstration of the role of CD73 in breast cancer may lead to new targeted therapies for breast cancer.

Effects of ecto-5'-nucleotidase on human breast cancer cell growth in vitro and in vivo.

Ecto-5'-nucleotidase (CD73) is an essential enzyme that generates adenosine, an essential molecule for cell growth. CD73 increases significantly in many breast cancers. In this study, alpha,beta-methylene adenosine-5'-diphosphate (APCP), a specific CD73 inhibitor was used to block the hydrolase's activity. Effects of CD73 were examined on human breast cancer cells MDA-MB-231 in culture for proliferation, cell cycle progression, and apoptosis before and after APCP treatment. The in vivo effect of CD73 was examined on MDA-MB-231 tumor xenograft growth in nude mice. Cell growth curve, cell cycle and apoptosis were observed with MTT assays and flow cytometry, respectively. Microvessel density (MVD) and lymph vessel density (LVD) of implanted tumor tissues was analyzed by immunohistochemistry for CD31 and VEGFR-3 staining respectively. Our results showed that APCP inhibited MDA-MB-231 viability in a dose-dependent manner. APCP (12 microM) increased the percentage of G0/G1 phase cells from 49.75 to 59.16% while it decreased S phase and G2/M cells from 24.85 and 18.65% to 21.65 and 12.55%, respectively. The percentages of early and late apoptotic cells were also decreased after APCP treatment. However, APCP treatment did not affect the percentage of normal cells. Xenograft of MDA-MB-231 cells in the APCP treatment group had lower volume and weight than those of control group (2.70+/-1.14 vs 1.41+/-0.39 cm(3) and 2.7+/-0.5 vs 1.3+/-0.2 g), accompanied with less vessel formation with a MVD of 5+/-1 compared to the control group's 10+/-2 and an LVD of 4+1 vs 7+2. Our results suggest that CD73 may promote tumor growth and serve as a marker of breast cancer progression.

Gene expression profile analysis of an isogenic tumour metastasis model reveals a functional role for oncogene AF1Q in breast cancer metastasis.

To study the molecular mechanisms underlying breast cancer metastasis, gene expression profile analysis was performed on two well-established breast cancer cell lines with high and low metastatic potentials: MDA-MB-435HM and MDA-MB-435LM. The analysis was conducted using cDNA microarrays containing 8000 genes. Of 60 differentially expressed genes, ALL1-fused gene from chromosome 1q (AF1Q), a putative oncogene not described previously in breast cancer, was identified and found to be over-expressed in MDA-MB-435HM cells compared with MDA-MB-435LM cells. The results indicate that AF1Q may play an important role in breast cancer metastasis. To test this hypothesis, we generated an AF1Q high-expression cell line by stable transfection of AF1Q cDNA into MDA-MB-435LM cells. Results showed that over-expression of AF1Q led to a marked increase in the invasive and metastatic potential of MDA-MB-435LM cells in vitro and in vivo, accompanied by the up-regulation of matrix metalloproteinase-2 (MMP-2), MMP-9, transcription factor Ets-1, and RhoC expression in both mRNA and protein levels. Consistent with this observation, reduced AF1Q expression in MDA-MB-435HM cells by small interfering RNA (siRNA) resulted in a significant decrease in the invasive potential of MDA-MB-435HM cells in vitro and in the protein expression of MMP-2, MMP-9, Ets-1, and RhoC, compared with either parental or non-silencing control cells. These data provide functional evidence that oncogene AF1Q may be a novel mediator of metastasis promotion in human breast cancer through regulation of the MMP pathway and RhoC expression.

Impaired upregulation of keratinocyte growth factor in injured lungs induced by Pseudomonas aeruginosa in immunosuppressed rats.

BACKGROUND: The number of immunosuppressed patients has increased in the past decades. Among them Pseudomonas aeruginosa (P. aeruginosa) is one of the leading bacteria for pneumonia that are associated with poor prognosis. However, the pathogenesis of P. aeruginosa pneumonia in immunosuppressed patients is not understood completely. Previous reports showed keratinocyte growth factor (KGF) is associated with lung injury in immunocompetent hosts. In this study, we investigated the different reactions of lung injury, lung pathology and KGF expressions in P. aeruginosa pneumonia between immunosuppressed and immunocompetent rats. METHODS: Immunosuppression of male rats was induced by injecting immunosuppressive subcutaneously. Pneumonia was established by instilling P. aeruginous tracheally. The immunocompetent rats were the control group. Survival rate, lung histopathology, pulmonary permeability and oedema, KGF mRNA and protein expressions in lungs of both groups were investigated. RESULTS: The survival rate of immunosuppressed group was lower than that of immunocompetent group (33.3% vs 83.3%). After exposure to bacteria, pulmonary permeability and wet/dry ratio in immunosuppressed group were higher than those in immunocompetent group. Pulmonary congestion and haemorrhage were more intensive in immunosuppressed group compared to immunocompetent group. Apoptosis and necrosis were also observed in infected lungs of immunosuppressed rats. Although we detected KGF expressions in lungs of both groups after infection, the expressions of KGF protein and mRNA gene in immunosuppressed group were much lower than in immunocompetent group. CONCLUSIONS: Compared with immunocompetent group, there was more intensive lung injury in immunosuppressed group. Severe lung injury may contribute to the poor prognosis of pneumonia. KGF expressions of pneumonia in immunosuppressed rats were less than those in immunocompetent ones.

[Downregulation of Duffy antigen receptor for chemokine (DARC) is associated with lymph node metastasis in human breast cancer].

OBJECTIVE: To analyze the relationship between Duffy antigen receptor for chemokines (DARC) and the metastasis potential in human breast cancer. METHODS Breast cancer tissue sections from 75 patients, grouped according to the local lymph node status were examined immunohistochemically for protein level of DARC. Microvessel density (MVD) was counted by endothelial cells immunostained using anti-CD34 antibody. RESULTS: Strong positive DARC immunostaining in lymph node negative and positive groups was detected in 31 cases (81.6%) and 18 cases (48.6%), respectively (P < 0.01). MVD was (35.67 +/- 17.96)/HP and (53.38 +/- 20.29)/HP in DARC strong positive and less positive cases (P < 0.01). In those patients with lung, bone, hepatic distant metastasis (13 cases), 9 cases (69.2%) were DARC less positive, 4 cases (30.8%) were DARC strong positive. The correlation coefficient was -0.412 between DARC expression and MVD and the corresponding value was -0.346 between DARC expression and lymph node status and -0.333 between DARC expression and distant metastasis in breast cancer. CONCLUSION: DARC may play a negative role in the process of neoangiogenesis, and probably has an association with the lymph node status.