Gen Hong Di

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.

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.

UHRF1 is associated with epigenetic silencing of BRCA1 in sporadic breast cancer

BRCA1 is closely related to the pathogenesis of breast cancer, BRCA1 mRNA is reduced in sporadic breast cancer cells despite the lack of mutations. In the present report, we found that overexpression of UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) was closely related to DNA methylation, deacetylation, and methylation of histones, recruitment of an inhibiting transcriptional complex on the BRCA1 promoter in sporadic breast cancer. Overexpression of UHRF1 induced deacetylation of histones H3 and H4, which was facilitated by recruitment of histone deacetylase1 (HDAC1) to the BRCA1 promoter. Loss of acetylation was accompanied by loss of binding of the key transcription factors MyoD, CBP, and p300. UHRF1 also recruited histone lysine methyltransferase G9a to the BRCA1 promoter and histone 3 lysine 4 (H3K4) was demethylated, and histone 3 lysine 9 (H3K9) was methylated. Finally, overexpression of UHRF1 leaded to methylation of BRCA1 promoter by recruitment of DNMT1 to the BRCA1 promoter, locking in marked suppression of BRCA1. It is the first to describe that UHRF1 is responsible for regulating BRCA1 transcription by inducing DNA methylation, histone modifications, and recruitment of transcriptional complex on the BRCA1 promoter, UHRF1 is a new bio-marker in sporadic breast cancer.

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)

A functional polymorphism in the promoter region of GSTM1 implies a complex role for GSTM1 in breast cancer.

Although a number of studies have been conducted to address the relation between a gene deletion polymorphism of glutathione S‐transferase M1 (GSTM1) and breast cancer, no definite conclusion has been reached and no clear risk pattern has yet to emerge for GSTM1. We first conducted case‐control studies that included 1920 subjects using a genotyping method allowing the definition of GSTM1‐null (–/–), homozygous wild‐type (+/+), and heterozygous (+/—) genotypes. The results show that GSTM1–/– confers an increased risk for breast cancer development compared with that in GSTM1‐present individuals (+/+ and +/–), which was subsequently confirmed by a meta‐analysis of all of the 41 relevant studies (odds ratio: 1.10, P<0.001). Unexpectedly, we found that GSTM1–/– is also a risk genotype compared with GSTM1–/–. Furthermore, we identified a functional polymorphism in the GSTM1 promoter region associated with breast cancer. The variant allele modifies DNA binding to the AP‐2α transcription factor, resulting in reduced promoter activity and mRNA expression. However, this low‐activity allele is associated with reduced breast cancer risk. It seems that ~60–70% expression from one allele of GSTM1 could suffice for protection against breast cancer;null activity and overactivity of GSTM1 are both disadvantageous. These results indicate a U‐shaped association of GSTM1 with breast cancer, which challenges the linear gene‐dosage effect of GSTM1 that was previously proposed. We recommend that a more complicated role for GSTM1 should be considered in breast cancer risk prediction.—Yu, K.D., Di, G.‐H., Fan, L., Wu, J., Hu, Z., Shen, Z.‐Z., Huang, W., Shao, Z.‐M. A functional polymorphism in the promoter region of GSTM1 implies a complex role for GSTM1 in breast cancer. FASEBJ. 23, 2274–2287 (2009)

PA-MSHA inhibits proliferation and induces apoptosis through the up-regulation and activation of caspases in the human breast cancer cell lines.

To investigate the effects of PA‐MSHA (Pseudomonas aeruginosa‐mannose sensitive hemagglutinin) on inhibiting proliferation of breast cancer cell lines and to explore its mechanisms of action in human breast cancer cells. MCF‐10A, MCF‐7, MDA‐MB‐468, and MDA‐MB‐231HM cells were treated with PA‐MSHA or PA (Heat‐killed P. aeruginosa) at different concentrations and different times. Changes of cell super‐microstructure were observed by transmission electron microscopy. Cell cycle distribution and apoptosis induced by PA‐MSHA were measured by flow cytometry (FCM) with PI staining, ANNEXIN V‐FITC staining and Hoechst33258 staining under fluorescence microscopy. Western blot was used to evaluate the expression level of apoptosis‐related molecules. A time‐dependent and concentration‐dependent cytotoxic effect of PA‐MSHA was observed in MDA‐MB‐468 and MDA‐MB‐231HM cells but not in MCF‐10A or MCF‐7 cells. The advent of PA‐MSHA changed cell morphology, that is to say, increases in autophagosomes, and vacuoles in the cytoplasm could also be observed. FCM with PI staining, ANNEXIN V‐FITC and Hoechst33258 staining showed that the different concentrations of PA‐MSHA could all induce the apoptosis and G0–G1 cell cycle arrest of breast cancer cells. Cleaved caspase 3, 8, 9, and Fas protein expression levels were strongly associated with an increase in apoptosis of the breast cancer cells. There was a direct relationship with increased concentrations of PA‐MSHA but not of PA. Completely different from PA, PA‐MSHA may impart antiproliferative effects against breast cancer cells by inducing apoptosis mediated by at least a death receptor‐related cell apoptosis signal pathway, and affecting the cell cycle regulation machinery. J. Cell. Biochem. 108: 195–206, 2009.

Estrogen Receptor (ER) β or p53 Attenuates ERα-mediated Transcriptional Activation on the BRCA2 Promoter

BRCA2 is closely related to the pathogenesis of breast cancer. In the present study, we found that estrogen can activate BRCA2 transcription, which is estrogen receptor (ER) α-dependent. During estrogen treatment, ERα interacted with CREB-binding protein/p300, p68/p72, and MyoD and formed an activating transcriptional complex that could bind to many Sp1 sites on the BRCA2 promoter and activate its transcription by inducing histone acetylations. MyoD is a new component of ERα complex. ERβ or p53 attenuated ERα-mediated transcriptional activation by preventing the recruitment of ERα transcriptional complex and histone acetylations on the BRCA2 promoter. ERβ interacted with ERα and CREB-binding protein/p300 and formed a weak activating transcriptional complex that competed for binding to Sp1 sites with ERα transcriptional complex and slightly attenuated BRCA2 transcription. Different from ERβ, p53 interacted with HDAC1 and CtBP1 and formed an inhibiting transcriptional complex that could compete for binding to Sp1 sites with ERα transcriptional complex and inhibit BRCA2 transcription more significantly.

Functional polymorphisms, altered gene expression and genetic association link NRH:quinone oxidoreductase 2 to breast cancer with wild-type p53

We hypothesized that NRH:quinone oxidoreductase 2 (NQO2) is a candidate susceptibility gene for breast cancer because of its known enzymatic activity on estrogen-derived quinones and its ability to stabilize p53. We performed case-control studies to investigate the contributions of genetic variants/haplotypes of the NQO2 gene to breast cancer risk. In the first hospital-based study (n = 1604), we observed significant associations between the incidence of breast cancer and a 29 bp-insertion/deletion polymorphism (29 bp-I/D) and the rs2071002 (+237A>C) polymorphism, both of which are located within the NQO2 promoter region. Decreased risk was associated with the D-allele of 29 bp-I/D [odds ratio (OR), 0.76; P = 0.0027] and the +237C-allele of rs2071002 (OR, 0.80; P = 0.0031). Specifically, the susceptibility variants within NQO2 were notably associated with breast carcinomas with wild-type p53 (the most significant P-value: 3.3 x 10(-6)). The associations were successfully replicated in an independent population set (familial/early-onset breast cancer cases and community-based controls, n = 1442). The combined P-values of the two studies (n = 3046) are 3.8 x 10(-7) for 29 bp-I/D and 2.3 x 10(-6) for rs2071002. Furthermore, we revealed potential mechanisms of pathogenesis of the two susceptibility polymorphisms. Previous work has demonstrated that the risk-allele I-29 of 29 bp-I/D introduces transcriptional-repressor Sp3 binding sites. Using promoter reporter-gene assays and electrophoretic-mobility-shift assays, our present work demonstrated that the other risk-allele, +237A-allele of rs2071002, abolishes a transcriptional-activator Sp1 binding site. Furthermore, an ex vivo study showed that normal breast tissues harboring protective genotypes expressed significantly higher levels of NQO2 mRNA than those in normal breast tissues harboring risk genotypes. Taken together, the data presented here strongly suggest that NQO2 is a susceptibility gene for breast carcinogenesis.

Breast cancer in Chinese elderly women: Pathological and clinical characteristics and factors influencing treatment patterns

The aims of this study are to describe tumor characteristics and treatment patterns of elder breast cancer patients and to determine the factors influencing local and systemic treatments. This retrospective cohort included 866 patients (>or=60 years) referred for surgery between January 2002 and December 2006. The patients were divided into four groups according to age. Elderly women had larger tumors at diagnosis with more mucinous carcinomas, more estrogen/progesterone-positive, lower Ki-67 labeling indices and less c-erbB2 positive tumors. Comorbidities were more often recorded for older patients. They were more likely to undergo simple mastectomy or breast-conserving surgery, less likely to receive adjuvant chemotherapy and radiotherapy, compared with their younger counterparts. Multinomial and binary logistic regression showed that age was independently associated with local and systemic treatments. Our data suggest that the tumors of elderly patients are biologically more favorable, and elderly women appear to receive less aggressive treatments.