Zhen Zhou Shen

Curcumin exerts multiple suppressive effects on human breast carcinoma cells

In our study, we present experimental evidence suggesting that curcumin exerts multiple different suppressive effects on human breast carcinoma cells in vitro. Our experiments demonstrate that curcumins antiproliferative effects are estrogen dependent in ER (estrogen receptor)‐positive MCF‐7 cells, being more pronounced in estrogen‐containing media and in the presence of exogenous 17‐β estradiol. Curcumin inhibits the expression of ER downstream genes including pS2 and TGF‐β (transforming growth factor) in ER‐positive MCF‐7 cells, and this inhibition is also dependent on the presence of estrogen. Curcumin also decreases ERE (estrogen responsive element)‐CAT activities induced by 17‐β estradiol. In addition, we demonstrate that curcumin exerts strong antiinvasive effects in vitro that are not estrogen dependent in the ER‐negative MDA‐MB‐231 breast cancer cells. These antiinvasive effects appear to be mediated through the downregulation of MMP‐2 (matrix metalloproteinase) and the upregulation of TIMP‐1 (tissue inhibitor of metalloproteinase), 2 common effector molecules that have been implicated in regulating tumor cell invasion. Our study also demonstrates that curcumin inhibits the transcript levels of 2 major angiogenesis factors VEGF (vascular endothelial growth factor) and b‐FGF (basic fibroblast growth factor) mainly in ER‐negative MDA‐MB‐231 cells.

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.

p21/waf1/cip1 and mdm-2 expression in breast carcinoma patients as related to prognosis.

p21/waf1/cip1 and mdm‐2 are downstream effectors of p53. p21 plays a major role in negatively regulating cell‐cycle progression, while mdm‐2 inhibits p53 effects, and its role has been implicated in oncogenesis. In this study, we investigated the expression profiles of p21, mdm‐2 and p53 in human breast‐carcinoma tissues. The aim was to determine whether a correlation exists between the expression profiles of these markers and tumor differentiation, ER status and prognosis. We studied tumor specimens obtained from 106 patients and found a highly significant association among low histology grade, p53 over‐expression, high mdm‐2 expression and lack of p21 expression. Our studies also demonstrate that, in human breast cancer, low levels of p21 and higher mdm‐2 levels directly correlate with the onset of lymph‐node metastases and shortened patient survival. Furthermore, the expression profiles of p21, mdm‐2 and p53 were independently correlated with patient survival. Int. J. Cancer 74:529–534, 1997.

Enhanced Expression of Rab27A Gene by Breast Cancer Cells Promoting Invasiveness and the Metastasis Potential by Secretion of Insulin-Like Growth Factor-II

In addition to the functions of transporting melanosome in melanocytes and releasing contents of lytic granules in CTLs, Rab27A was recently shown to be involved in exocytosis of insulin and chromaffin granules in endocrine cells; it was also reported to be expressed in an exceptionally broad range of specialized secretory cells. As autocrine and paracrine cytokines are essential for invasion and metastasis in some solid tumors, blocking them may be an effective strategy to prevent tumor dissemination. In the present study, we show that Rab27A is associated with invasive and metastatic potentials of human breast cancer cells. The overexpression of Rab27A protein redistributed the cell cycle and increased the invasive and metastatic abilities in breast cancer cells both in vitro and in vivo. We also certified that Rab27A conferred the invasive and metastatic phenotypes on breast cancer cells by promoting the secretion of insulin-like growth factor-II (IGF-II), which regulates the expression of p16, vascular endothelial growth factor, matrix metalloproteinase-9, cathepsin D, cyclin D1, and urokinase-type plasminogen activator. These data provide functional evidence that Rab27A acts as a novel mediator of invasion and metastasis promotion in human breast cancer cells, at least in part, through regulating the secretion of IGF-II, suggesting that synergistic suppression of Rab27A and IGF-II activities holds a promise for preventing breast cancer invasion and metastasis. (Mol Cancer Res 2008;6(3):372–82)

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)

ERβ exerts multiple stimulative effects on human breast carcinoma cells

Recent studies of ERs in breast cancer have demonstrated the existence of ERβ in addition to ERα. Some clinical data indicated that ERβ had prognostic value for patients survival, which suggested that ERβ plays a key role in breast cancer development and metastasis. To test this hypothesis, we generated an ERβ high-expression cell line by reintroduced human ERβ cDNA into MDA-MB-435 cells. We demonstrated that ERβ exerted multiple tumor-stimulative effects on human breast carcinoma cells both in vivo and in vitro. In in vitro studies, ERβ was able to increase the proliferation and invasion of MDA-MB-435 cells significantly, while these effects were totally estradiol independent. Also, this stimulation was characterized by downregulation of p21 and upregulation of MMP-9, as well as transcriptional factor Est-1. In in vivo studies, we also demonstrated that ERβ-transfected MDA-MB-435 cells grew much faster and had more pulmonary metastasis than mock or wild-type cells in nude mice. In ERβ-transfected MDA-MB-435 xenografts, ERβ caused significant reduction in p21 protein levels. Similar effects of ERβ on MMP-9 and Ets-1 expression noted in vitro studies were also observed in the in vivo studies. These in vitro and in vivo studies indicated that ERβ exerted multiple stimulative effects on breast cancer development and metastasis.

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.

MicroRNA-200a Promotes Anoikis Resistance and Metastasis by Targeting YAP1 in Human Breast Cancer

Purpose: The process of metastases involves the dissociation of cells from the primary tumor, penetration into the basement membrane, invasion, and exiting from the vasculature to seed and colonize distant tissues. miR-200a is involved in this multistep metastatic cascade. This study aimed to test the hypothesis that miR-200a promotes metastasis through increased anoikis resistance in breast cancer. Experimental Design: Breast cancer cells transfected with mimic or inhibitor for miR-200a were assayed for anoikis in vitro. miR-200a expression was assessed by quantitative real-time PCR (qRT-PCR). Luciferase assays, colony formation assays, and animal studies were conducted to identify the targets of miR-200a and the mechanism by which it promotes anoikis resistance. Results: We found that overexpression of miR-200a promotes whereas inhibition of miR-200a suppresses anoikis resistance in breast cancer cells. We identified Yes-associated protein 1 (YAP1) as a novel target of miR-200a. Our data showed that targeting of YAP1 by miR-200a resulted in decreased expression of proapoptotic proteins, which leads to anoikis resistance. Overexpression of miR-200a protected tumor cells from anoikis and promoted metastases in vivo. Furthermore, knockdown of YAP1 phenocopied the effects of miR-200a overexpression, whereas restoration of YAP1 in miR-200a overexpressed breast cancer cells reversed the effects of miR-200a on anoikis and metastasis. Remarkably, we found that YAP1 expression was inversely correlated with miR-200a expression in breast cancer clinical specimens, and miR-200a expression was associated with distant metastasis in patients with breast cancer. Conclusions: Our data suggest that miR-200a functions as anoikis suppressor and contributes to metastasis in breast cancer. Clin Cancer Res; 19(6); 1389–99. ©2013 AACR.

Development and trends of surgical modalities for breast cancer in China: a review of 16-year data.

BackgroundSurgery is the most important treatment for nonmetastatic breast cancer; however, the utilization of modern surgical techniques in management of breast cancer in mainland China has not been reported.MethodsThe medical records of 5887 consecutive breast cancer patients treated surgically in the past 16 years were reviewed retrospectively; the utilization of different surgical modalities and associated clinical outcomes were analyzed.ResultsMedian age of all patients was 50 (range 16–92). About 1015 patients were staged as 0–I, 3569 stage II, 517 stage III, and 786 cases could not be staged. Extensive radical mastectomy (ERM), radical mastectomy (RM), modified radical mastectomy (MRM), simple mastectomy (SM), and breast-conserving surgery (BCS) were used in 8%, 27.2%, 55.7%, 1.5%, and 6.3% of patients, respectively. In addition, 1.3% of patients received breast reconstruction. The proportion of early-stage breast cancer increased, and the surgery patterns varied. MRM gradually replaced ERM and RM. The prevalence of BCS began to increase from the mid-1990s and currently represents about 12%. The prevalence of reconstruction also increased and now accounts for 5%. Age, pathologic pattern, and TNM staging affected the choice of surgery modalities markedly. Although patients receiving RM/ERM had worse survival than those receiving BCS/MRM, the survival outcomes of these four groups were similar in the early-stage population.ConclusionsMRM remains the most-used surgical modality in operable breast cancer, although the utilization of BCS for early-stage disease has increased rapidly in last decade. Reconstruction following mastectomy as an alternative to BCS is available. Breast-conserving therapy (BCT) and MRM provide similar local controls and long-term survival for breast cancer. Selection of appropriate candidates for a certain surgery requires an assessment of the patient’s age and clinical and pathological characteristics of the tumor.

Enhanced expression of LKB1 in breast cancer cells attenuates angiogenesis, invasion, and metastatic potential

LKB1 (also known as STK11) is a recently identified tumor suppressor gene whose mutation can lead to Peutz-Jeghers syndrome, which is characterized by gastrointestinal polyps and cancers of different organ systems. Approximately 30% of sporadic breast cancer samples express low levels of LKB1. This suggests that the LKB1 gene may be related to the tumorigenesis of breast cancer. We reintroduced LKB1 into MDA-MB-435 breast cancer cells that lack the LKB1 gene to investigate how overexpression of LKB1 affects tumor invasiveness and metastasis. Overexpression of the LKB1 protein in breast cancer cells resulted in significant inhibition of in vitro invasion. In vivo, LKB1 expression reduced tumor growth in the mammary fat pad, microvessel density, and lung metastasis. LKB1 overexpression was associated with down-regulation of matrix metalloproteinase-2, matrix metalloproteinase-9, vascular endothelial growth factor, and basic fibroblast growth factor mRNA and protein levels. Overexpression of the LKB1 protein in human breast cancer is significantly associated with a decrease in microvessel density. Our results indicate that LKB1 plays a negative regulatory role in human breast cancer, a finding that may lead to a new therapeutic strategy. (Mol Cancer Res 2006;4(11):843–9)