Zhou Luo Ou

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

IntroductionThe 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.MethodsExpression 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.ResultsOverexpression 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.ConclusionOverexpression 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.

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. (Mol Cancer Res 2008;6(8):1276–88)

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-1β, tumor necrosis factor-α, and IFN-γ. 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.

Gemcitabine resistance in breast cancer cells regulated by PI3K/AKT-mediated cellular proliferation exerts negative feedback via the MEK/MAPK and mTOR pathways.

Chemoresistance is a major cause of cancer treatment failure and leads to a reduction in the survival rate of cancer patients. Phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) and mitogen-activated protein kinase (MAPK) pathways are aberrantly activated in many malignant tumors, including breast cancer, which may indicate an association with breast cancer chemoresistance. In this study, we generated a chemoresistant human breast cancer cell line, MDA-MB-231/gemcitabine (simplified hereafter as “231/Gem”), from MDA-MB-231 human breast cancer cells. Flow cytometry studies revealed that with the same treatment concentration of gemcitabine, 231/Gem cells displayed more robust resistance to gemcitabine, which was reflected by fewer apoptotic cells and enhanced percentage of S-phase cells. Through the use of inverted microscopy, Cell Counting Kit-8, and Transwell assays, we found that compared with parental 231 cells, 231/Gem cells displayed more morphologic projections, enhanced cell proliferative ability, and improved cell migration and invasion. Mechanistic studies revealed that the PI3K/AKT/mTOR and mitogen-activated protein kinase kinase (MEK)/MAPK signaling pathways were activated through elevated expression of phosphorylated (p)-extracellular signal-regulated kinase (ERK), p-AKT, mTOR, p-mTOR, p-P70S6K, and reduced expression of p-P38 and LC3-II (the marker of autophagy) in 231/Gem in comparison to control cells. However, there was no change in the expression of Cyclin D1 and p-adenosine monophosphate-activated protein kinase (AMPK). In culture, inhibitors of PI3K/AKT and mTOR, but not of MEK/MAPK, could reverse the enhanced proliferative ability of 231/Gem cells. Western blot analysis showed that treatment with a PI3K/AKT inhibitor decreased the expression levels of p-AKT, p-MEK, p-mTOR, and p-P70S6K; however, treatments with either MEK/MAPK or mTOR inhibitor significantly increased p-AKT expression. Thus, our data suggest that gemcitabine resistance in breast cancer cells is mainly mediated by activation of the PI3K/AKT signaling pathway. This occurs through elevated expression of p-AKT protein to promote cell proliferation and is negatively regulated by the MEK/MAPK and mTOR pathways.

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.

Effect of Genetic Variants in Two Chemokine Decoy Receptor Genes, DARC and CCBP2, on Metastatic Potential of Breast Cancer

The inhibitory effect of two chemokine decoy receptors (CDRs), DARC and D6, on breast cancer metastasis is mainly due to their ability to sequester pro-malignant chemokines. We hypothesized that genetic variants in the DARC and CCBP2 (encoding D6) genes may be associated with breast cancer progression. In the present study, we evaluated the genetic contributions of DARC and CCBP2 to metastatic potential, indicated by lymph node metastasis (LNM). Ten single-nucleotide polymorphisms (SNPs) (potentially functional SNPs and block-based tagging SNPs) in DARC and CCBP2 were genotyped in 785 breast cancer patients who had negative lymph nodes and 678 patients with positive lymph nodes. Two non-synonymous SNPs, rs12075 (G42D) in DARC and rs2228468 (S373Y) in CCBP2, were observed to be associated with LNM in univariate analysis and remained significant after adjustment for conventional clinical risk factors, with odds ratios (ORs) of 0.54 (95% confidence interval [CI], 0.37 to 0.79) and 0.78 (95% CI, 0.62 to 0.98), respectively. Additional functional experiments revealed that both of these significant SNPs could affect metastasis of breast cancer in xenograft models by differentially altering the chemokine sequestration ability of their corresponding proteins. Furthermore, heterozygous GD genotype of G42D on human erythrocytes had a significantly stronger chemokine sequestration ability than homozygous GG of G42D ex vivo. Our data suggest that the genetic variants in the CDR genes are probably associated with the varied metastatic potential of breast cancer. The underlying mechanism, though it needs to be further investigated, may be that CDR variants could affect the chemokine sequestration ability of CDR proteins.

CCL28 promotes breast cancer growth and metastasis through MAPK-mediated cellular anti-apoptosis and pro-metastasis

Breast cancer is one of the most commonly diagnosed cancers worldwide and the second leading cause of cancer-related deaths among females. CCL28 (mucosa-associated epithelial chemokine, MEC), a CC subfamily chemokine, has been well studied in the process of inflammation, and recently increasing evidence indicates that CCL28 is related to tumor progression. However, little is known concerning its function in breast cancer. In the present study, we generated a CCL28-overexpressing breast cancer cell line MDA-MB-231HM/CCL28 from parental MDA-MB-231HM cells. We found that overexpression of CCL28 promoted cell proliferation and tumor formation, and also enhanced migration, invasion and metastasis both in vitro and in vivo. Mechanistic studies revealed that CCL28 mediated intracellular activation of the mitogen-activated protein kinase (MAPK) signaling pathway to promote breast cancer cell proliferation and metastasis by upregulating anti-apoptotic protein Bcl-2 and suppressing cell adhesion protein β-catenin. However, overexpression of CCL28 did not influence the expression of metastasis-related protein matrix metalloproteinase MMP2 and MMP9 and VEGF. Tissue sample analysis from animal models also indicated that overexpression of CCL28 was associated with enhanced pERK expression and reduced β-catenin expression in breast carcinomas. Thus, our results show for the first time that CCL28 contributes to breast cancer progression through the ERK/MAPK-mediated anti-apoptotic and metastatic signaling pathway. Antagonists of CCL28 and the MAPK signaling pathway may be used synergistically to treat breast cancer patients.

Anti-Cervical Cancer Role of Matrine, Oxymatrine and Sophora Flavescens Alkaloid Gels and its Mechanism

Background: Cervical cancer is one of the leading severe malignancies throughout the world. Sophra flavescens alkaloid (SFA) gels, a compound Traditional Chinese Medicine, has been clinically used in China for many years. Its individual active ingredients are matrine and oxymatrine, which has been showed that they can restrain primary tumorigenesis, while the underlying molecular mechanisms of SFA gels in cervical cancer cells remain unclear. Methods: To detect the effect of SFA gels and its active ingredients, CCK-8 assay and colony assay were used on cervical cancer cells proliferation. Transwell assay was used to detect cancer cell migration. Apoptosis and cell cycle arrest were used to detect whether SFA gels effect the cervical cancer cells proliferation. Western blot was used to detect whether SFA gels regulate the cervical cancer cells via the suppression of AKT/mTOR signaling pathway. Results: SFA gels can restrain cervical cancer cell proliferation, inhibit metastasis, induce cell cycle arrest in G2/M phase, induce cellular apoptosis through stimulation of Bax and E-cadherin, and suppression of Bcl-2, cyclin A, MMP2. Further study shows that SFA gels may regulate the cervical cancer cells via the suppression of AKT/mTOR signaling pathway. Conclusions: SFA gels, like its active ingredients, can restrain cervical cancer cells proliferation, suppress cervical cancer cell migration, induce the apoptosis and cell cycle arrest in cervical cancer cells. SFA gels may be a potential anti-tumor therapeutic agent for treating cervical cancer.

The role of the chemokine receptor XCR1 in breast cancer cells

Considerable attention has recently been paid to the application of chemokines to cancer immunotherapy due to their complex role in cell proliferation, invasion, metastasis, and tumorigenesis, which extends beyond the regulation of lymphocyte migration during immune responses. The expression and the function of the chemokine receptor XCR1 on breast cancer have remained elusive to date. In this study, the expressions of XCR1 mRNA were tested by quantitative real-time polymerase chain reaction in one breast epithelial cell line (MCF-10A) and nine breast cancer cell lines (MDA-MB-231, 231HM, 231BO, MDA-MB-468, MCF-7, T47D, Bcap-37, ZR-75-30, and SK-BR-3). We established XCR1-overexpressing breast cancer cell line MDA-MB-231 (231/XCR1) in XCR1 low expression cell line MDA-MB-231 (231). The ability of proliferation, invasion, and metastasis was measured by CCK8, plate cloning formation, and transwell analysis, respectively, in XCR1-overexpressing breast cancer cell lines (231/XCR1) and their parental cell line MDA-MB-231/Vector (simplified as “231/Vector”); 5×106/100 μL cells were inoculated in mammary fat pad of BALB/c nude mice. There were six BALB/c nude mice in the experimental group and control group. Protein expression was analyzed by cell immunofluorescence and Western blot. The growth of XCR1-overexpressing human breast cancer cell line MDA-MB-231 in vitro was restrained and tumorigenesis in vivo was also extenuated, its mechanism may involve in the inhibition of MAPK and PI3K/AKT/mTOR signaling pathway, but increase in LC3 expression. However, the overexpression of XCR1 in human breast cancer cell line MDA-MB-231 in vitro can promote the migration and invasion partially due to decreasing the protein level of β-catenin. Therefore, XCR1 can affect the biological characteristics of some special breast cancer cells through complex signal transduction pathway.

The human chemokine receptor CCRL2 suppresses chemotaxis and invasion by blocking CCL2-induced phosphorylation of p38 MAPK in human breast cancer cells

Abstract The human chemokine receptor CCRL2 is a member of the atypical chemokine receptor family. CCRL2 is unable to couple with G-proteins and fails to induce classical chemokine signaling for the highly conserved DRYLAIV motif essential for signaling has been changed to QRYLVFL. We investigated whether CCRL2 is involved in the chemotaxis, invasion, and proliferation of human breast cancer cells. Firstly, expression of CCRL2 was determined in six breast cancer cell lines by real-time RT-PCR and Western blot. Then, we established stable cell lines overexpressing CCRL2 to explore the function of CCRL2 in chemotaxis and invasion by transwell assays, and the signaling downstream was further investigated. The effect of CCRL2 on proliferation was detected by colony formation assays and tumor xenograft study. We found that stable overexpression of CCRL2 in MDA-MB-231 and BT-549 cells attenuated the chemotaxis and invasion stimulated by its ligand CCL2. CCRL2 inhibits p38 MAPK (p38) phosphorylation and up-regulates the expression of E-cadherin. This effect was eliminated by the inhibitor of p38 MAPK. CCRL2 inhibited the growth of breast cancer cells in vitro and in vivo. Our results suggest that CCRL2 functions as a tumor suppressor in human breast cancer cells.