Table S3 - PDF file 67K, Univariate and multivariable Cox regression analysis of relapse-free survival (Cox proportional hazards regression model)

Curcumin, a biphenyl compound derived from rhizome, is a powerful anti-cancer agent. Emodin is an active component isolated from the root and rhizome of Rheum palmatum that has been widely used in traditional Chinese medicine for the treatment of various diseases. Currently, there are no studies examining the effect of curcumin in combination with emodin on tumor cell growth. In this study, we report for the first time that combined curcumin and emodin administration synergistically inhibits proliferation (MTT assay), survival (flow cytometry), and invasion (transwell migration assay) of breast cancer cells. Synergism is determined by the Chou–Talalay method. Moreover, we demonstrate that miR-34a is upregulated by curcumin and emodin. This microRNA helps mediate the anti-tumor effects of curcumin and emodin by downregulating Bcl-2 and Bmi-1. Our results not only provide insight into the mechanism of synergy between curcumin and emodin in breast cancer cells, but also suggest a new and potentially useful approach for breast cancer therapy.

Background Various studies have been searching for new tumor biomarkers for breast cancer for years. However, so far, few markers have been proved clinically useful except CA153. Based on knowledge that most adenocarcinomas including breast carcinoma expressed Cytokeratin19, the authors studied CK19-2G2,a novel fragment of cytokeratin19 shedding into serum in breast cancer patients. Patients and Methods The serum samples of four hundred and seventeen patients including three hundred and three (fifty-four DCIS and two hundred and forty-nine stage I-III) PBC patients and one hundred and fourteen MBC patients, eighty-one healthy controls and twenty-one breast benign disease patients were provided for measurement of CK19-2G2, CEA and CA153.The correlation between clinicopathological characters, prognosis and CK19-2G2 levels was further studied. Results The serum CK19-2G2 levels in breast cancer patients were significantly higher than that in healthy and benign controls. For breast cancer patients, CK19-2G2 levels in MBC were significantly higher than that in PBC patients. The sensitivities of CK19-2G2 for breast carcinoma are as high as CEA and CA153, and up to 71% in MBC patients. Serum CK19-2G2 levels (≥2 mU/mL) were associated with pathological stages, tumor size (≥2 cm), lymph node involvement, and HER2 status. Multivariate analysis revealed that high serum CK19-2G2 level was an independent factor for relapse (P = 0.029) and death (P = 0.040) in breast cancer patients. Conclusion Serum CK19-2G2 may be an independent indicator for prognosis and a candidate marker for monitoring metastasis in breast cancer.

The purpose of this study was to identify a mechanism related to miRNA pathway which plays a role in the anti-tumor effects of Diallyl disulfide. Alterations in miRNA expression were observed in Diallyl disulfide-treated MGC-803 cells, including up-regulation of miR-200b and miR-22 expression. Furthermore, Wnt-1 was identified as a target of both miR-200b and miR-22. MiR-200b and miR-22 not only synergistically inhibited gastric cancer growth, but also enhanced the antitumor effect of Diallyl disulfide both in vitro and in vivo. It indicated that miR-200b and miR-22 may serve as potential gene therapy and enhance Diallyl disulfide antitumor effects.

Abstract Purpose: The purpose of this study was to investigate the clinicopathologic significance and potential role of miR-200b and miR-200c in the development and progression of gastric cancer. Experimental Design: We examined miR-200b and miR-200c expression in 36 paired normal and stomach tumor specimens, as well as gastric cancer cell lines, by quantitative real-time PCR. In addition, miR-200b and miR-200c were detected by ISH using gastric cancer tissue microarrays, and the association between miR-200b and miR-200c levels and clinicopathologic factors and prognosis were analyzed. A luciferase assay was conducted for target evaluation. The functional effects of miR-200b and miR-200c on gastric cancer cells were validated by a cell proliferation assay and cell invasion and migration assays. Results: miR-200b and miR-200c were downregulated in the gastric cancer specimens and cell lines tested. miR-200b and miR-200c levels were significantly correlated with the clinical stage, T stage, lymph node metastasis, and survival of patients. Ectopic expression of miR-200b and miR-200c impaired cell growth and invasion. In addition, when overexpressed, miR-200b and miR-200c commonly directly targeted DNMT3A, DNMT3B, and SP1 (a transactivator of the DNMT1 gene), which resulted in marked reduction of the expression of DNA methyltransferases DNMT1, DNMT3A, and DNMT3B at the protein level. This effect, in turn, led to a decrease in global DNA methylation and reexpression of p16, RASS1A1, and E-cadherin via promoter DNA hypomethylation. Conclusion: Our findings suggest that miR-200b and miR-200c, as valuable markers of gastric cancer prognosis, may be a promising approach to human gastric cancer treatment. Clin Cancer Res; 19(20); 5602–12. ©2013 AACR.

MicroRNAs (miRNAs) have been documented as playing important roles in cancer development. In this study, we investigated the role of miR-124 in breast cancer and clarified the regulation of flotillin-1 (FLOT1) by miR-124.

There are few delivery agents that could deliver gene with high efficiency and low toxicity, especially for animal experiments. Therefore, creating vectors with good delivery efficiency and safety profile is a meaningful work. We have developed a self-assembled gene delivery system (XM001), which can more efficiently deliver DNA to multiple cell lines and breast tumor, as compared to commercial delivery agents. In addition, systemically administrated XM001–BikDD (BikDD is a mutant form of proapoptotic gene Bik) significantly inhibited the growth of human breast cancer cells and prolonged the life span in implanted nude mice. This study demonstrates that XM001 is an efficient and widespread transfection agent, which could be a promising tumor delivery vector for cancer targeted therapy.

Breast cancer is the most commonly malignancies in women. MicroRNAs are a family of small non-coding RNAs 18-25 nucleotides in length that post-transcriptionally modulate gene expression. MiR-26a has been reported as a tumor suppressor microRNA in breast cancer, which is attributed mainly to targeting of MTDH and EZH2, however, the expression profile and therapeutic potential of miR-26a is still unclear. Here we demonstrate that miR-26a is down-regulated in breast cancer cells and clinical specimens and its modulation in breast cancer cells regulates cell proliferation, colony formation, migration and apoptosis. MCL-1, an anti-apoptotic member of the Bcl-2 family, as novel targets of miR-26a was found to be in reverse correlation with ectopic expression of miR-26a and knockdown of MCL-1 phenocopied the effect of miR-26a in breast cancer cell lines. It was further explored that miR-26a increased sensitivity of breast cancer cells to paclitaxel in which MCL-1 was involved. Thus, miR-26a impacts on cell proliferation and migration of breast cancer by regulating several carcinogenesis-related processes, including a novel mechanism involving the targeting of MCL-1.

Background Compared with invasive breast cancer, breast cancer in situ (BCIS) is seldom life threatening. However, an increasing incidence has been observed in recent years over the world. The purpose of our study is to investigate the epidemiological, clinical and pathological profiles of BCIS in Chinese women from 1999-2008. Methods Four thousand and two hundred-eleven female breast cancer (BC) patients were enrolled in this hospital-based nation-wide and multi-center retrospective study. Patients were randomly selected from seven hospitals in seven representative geographical regions of China between 1999 and 2008. The epidemiological, clinical and pathological data were collected based on the designed case report form (CRF). Results There were one hundred and forty-three BCIS cases in four thousand and two hundred-eleven BC patients (3.4%). The mean age at diagnosis was 48.3 years and BCIS peaked in age group 40-49 yrs (39.9%). The most common subtype was ductal carcinoma in situ (DCIS) (88.0%). 53.8% were positive for estrogen receptor (ER). Human epidermal growth factor receptor 2 (HER2) positive status was observed in 23.8% of patients. All patients underwent surgeries and 14.7% of them had breast conservation therapies (BCT) (21/143), but 41.9% accepted chemotherapy (64/143). Much less patients underwent radiotherapy (16.0%, 23/143) and among patients who had BCT, 67% accepted radiotherapy (14/21). Endocrine therapy was taken in 44.1% patients (63/143). Conclusions The younger age of BCIS among Chinese women than Western countries and increasing number of cases pose a great challenge. BCT and endocrine therapy are under great needs.

Breast cancer is a major public health problem all over the world, and the current treatment strategies are not potent enough for some patients, especially those with triple-negative breast cancer. Therefore, novel and more effective treatments are critically needed. Of the current methods, targeted therapy, which not only retains cancer-specific expression but also limits toxicity, is a new strategy for treating cancers. In this study, we found that the human telomerase reverse transcriptase (hTERT; T) promoter also possesses high target specificity in breast cancer. Moreover, we developed a versatile T-based breast cancer–specific promoter VISA (VP16-Gal4-WPRE integrated systemic amplifier) composite (T-VISA) to target transgene expression in breast tumors, which has stronger activity comparable or higher than that of the cytomegalovirus promoter in cancer cells. Thereafter, targeted expression of BikDD (a mutant form of proapoptotic gene Bik) through the T-VISA platform in breast cancer initiated robust antitumor effects and prolonged survival in multiple xenograft and syngeneic orthotopic mouse models of breast tumors with virtually no toxicity in intact mice. Thus, these findings show that our T-VISA-BikDD nanoparticles effectively and safely eradicate breast cancer in vitro and in vivo and are worthy of development in clinical trials treating breast cancer. Mol Cancer Ther; 11(9); 1915–24. ©2012 AACR.

Several researches reported that overexpression of SIRT1 was associated with poor survival in several human cancers. However, some researches reported that SIRT1 had an antitumor potential. The definite role of SIRT1 is not clear now, and few studies have documented the value of SIRT1 in triple-negative breast cancer (TNBC). Therefore, the aim of this study is to evaluate the role of SIRT1 in TNBC and non-TNBC for prognosis. A total of 51 TNBC patients and 83 non-TNBC patients who were diagnosed from October 2001 to September 2006 were involved in this study. Immunohistochemical staining for SIRT1 and p53 on tissue microarrays were used. Expression of SIRT1 was seen in 55 % of TNBC patients and 53 % of non-TNBC patients. Expression of SIRT1 was associated with lymph nodes status, stage, distant metastatic relapse, and p53 status in TNBC patients. Expression of SIRT1 in non-TNBC patients was significantly correlated with lymph nodes status, age, stage, distant metastatic relapse, PR status, and p53 status. SIRT1+ group was associated with shorter DFS and OS compared with SIRT1- group in TNBC, non-TNBC, and overall breast cancer patients, according to univariate Cox regression analysis. Our study provides evidence that expression of SIRT is associated with worse prognosis in TNBC and non-TNBC and SIRT1 could be a potential therapeutic target in breast cancer.

Purpose High serum human epidermal growth factors receptor-2 (HER2) extracellular domain (ECD) has been identified as an independent prognostic indicator of poor prognosis in metastatic breast cancer. However, its prognostic value in primary operable breast cancer was still controversial. We aim to investigate the correlation between serum HER2 ECD levels and tissue HER2 status, the association between serum HER2 ECD levels and clinicopathological characteristics, and their impacts on disease-free survival (DFS) and overall survival (OS) in primary operable breast cancer. Methods Two hundred and fifty-two primary operable breast cancer patients pretreated from 2002 to 2009 in Sun Yat-Sen University Cancer Center were enrolled in this study. Serum HER2 ECD was measured by chemiluminescent assay, and tissue HER2 status was accessed by immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) assay. Results There was a significant correlation between serum HER2 ECD levels and HER2 tissue status ( P < 0.001, R = 0.36). High serum HER2 ECD levels (≥15 ng/mL) were significantly associated with age (≥35 years) ( P = 0.028), postmenopausal status ( P < 0.001), stage III ( P < 0.001), tumor size (≥2 cm) ( P < 0.001), lymph node involvement ( P < 0.001), negative estrogen receptor ( P = 0.005), and progesterone receptor status ( P = 0.001). Multivariate analysis showed that high serum HER2 ECD level was an independent prognostic factor of worse DFS ( P = 0.014) and OS ( P = 0.014) in primary operable breast cancer patients. Conclusion Serum HER2 ECD level can reflect tissue HER2 status and can be an independent prognostic indicator for primary operable breast cancer patients.

Breast cancer is a major public health problem all over the world, and the current treatment strategies are not potent enough for some patients, especially those with triple-negative breast cancer. Therefore, novel and more effective treatments are critically needed. Of the current methods, targeted therapy, which not only retains cancer-specific expression but also limits toxicity, is a new strategy for treating cancers. In this study, we found that the human telomerase reverse transcriptase (hTERT; T) promoter also possesses high target specificity in breast cancer. Moreover, we developed a versatile T-based breast cancer–specific promoter VISA (VP16-Gal4-WPRE integrated systemic amplifier) composite (T-VISA) to target transgene expression in breast tumors, which has stronger activity comparable or higher than that of the cytomegalovirus promoter in cancer cells. Thereafter, targeted expression of BikDD (a mutant form of proapoptotic gene Bik) through the T-VISA platform in breast cancer initiated robust antitumor effects and prolonged survival in multiple xenograft and syngeneic orthotopic mouse models of breast tumors with virtually no toxicity in intact mice. Thus, these findings show that our T-VISA-BikDD nanoparticles effectively and safely eradicate breast cancer in vitro and in vivo and are worthy of development in clinical trials treating breast cancer. Mol Cancer Ther; 11(9); 1915–24. 2012 AACR.

Dear Editor: Breast cancer is a multifactorial disease. It is the first cause of mortality per cancer for the woman in the world (Jemal et al., 2011). The implication of specific genes, such as BRCA1 and BRCA2 tumor suppressor genes, has been shown in mammary carcinogenesis. In sporadic breast cancers, specific modifications of BRCA1 and BRCA2 mRNA expression have been reported too (Bernard-Gallon et al., 1999; Bieche et al., 1999). Epigenetic modifications such as promoter hypermethylation of the oncosuppressor genes BRCA1 and BRCA2 can play a role in the oncogenesis of cancer (Esteller et al., 2000). Indeed, hypermethylation of the CpG islands in the promoters of these genes involve their inactivation and therefore a higher risk of developing a tumor (Baylin and Ohm, 2006; Moelans et al., 2011; Rahmatpanah et al., 2009). We have compared the DNA methylation rates of the BRCA1 and BRCA2 gene promoters using DNA isolated from blood samples from COSA (Cancer d'Ovaire et du Sein en Auvergne) patients suffering from breast or ovarian cancer and from a population of healthy women. To this aim, the QAMA method (Quantitative Analysis of Methylated Alleles) was used (Zeschnigk et al., 2004) and adapted to BRCA1 and BRCA2 genes (Bosviel et al., 2011). This method is based on bisulfite conversion of the nonmethylated cytosines and an analysis by qPCR using Taqman minor groove binder probes specific for methylated or nonmethylated target sites after conversion. Percentage of methylation is then obtained by calculating the difference between CT values of the methylated DNA targeting probe and the nonmethylated DNA targeting probe (ΔCT) and reporting the obtained values on a standard curve. In a previous work, we reported that BRCA1 methylation is significantly decreased in ovarian cancer by comparison with the control group. The comparison between the two different populations did not show any significant difference regarding BRCA2 methylation but exhibited a trend in the decrease of BRCA2 promoter methylation in peripheral blood DNA of sporadic ovarian cancer (Bosviel et al., 2011). Then we demonstrated a trend toward BRCA1 promoter hypermethylation in PBCs of sporadic breast cancer patients by comparison with controls (Bosviel et al., 2012). BRCA1 promoter methylation in PBCs corresponded to 47.1% with CI 95% [46.1; 48.1] in breast cancer patients and to 45.9% with CI 95% [45.0; 46.8] in controls. Association between methylation level and clinicopathological features were evaluated using statistical tests. BRCA1 promoter methylation in PBCs increased significantly in breast cancer patients by comparison with controls, with the age over 70 years old (p=0.022), in post menopausal status (p=0.013), with a BMI<20 (p=0.0095), or with a WHR≤76 .8 (p=0.0027). We also found an association of increased BRCA1 promoter methylation in PBCs with ACA/ACA genotype for the SNP Thr594Thr in ESR (estrogen receptor), known to be associated with breast cancer risk (p=0.092), due to the reduced presence of this genotype in this breast cancer case-control study. Within this study, the objective was to compare the methylation of the CpG islands present in the BRCA2 promoter in the same population of women suffering from breast cancer compared to the control population. In total, 873 breast samples belonging to COSA and 980 control samples were converted and the methylation rates measured. The complete database of this study is available as supplementary data (supplementary data are available online at www.liebertonline.com/omi). BRCA2 promoter methylation mean in PBCs is 16.9% (CI95% [16.3; 17.4]) in breast cancer patients and 16.2% (CI95% [15.7; 16.8]) in controls. The statistical analysis of the mean methylation rates obtained for the BRCA2 promoter did not reveal a significant difference (p=0.1) between the two populations (Fig. 1). FIG. 1. Average percentage of methylation of the promoter of BRCA2 in DNA from control patients and COSA (p=0.10). Significant differences in methylation rates between patients and healthy women were, however, obtained for different subclasses (Table 1). It reached 17.3% in breast cancer patients older than 70 years by comparison with control patients (14.7%) with p=0.016. In the subclass with an early menopause (before 48 years), the level was respectively 16.6% in breast cancer patients versus 15.4% in control patients (p=0.028). Table 1. Demographics of Breast Cancer Patients and Controls and Their Association with BRCA2 Promoter Methylation in Peripheral Blood Cells Then, breast cancer patients showing a normal BMI (Body Mass Index) [20–25[ exhibited a BRCA2 promoter methylation in PBCs of 17.6% versus 16.5% for control patients (p=0.019). Concerning the increase in the WHR (Waist-to-Hip Ratio) ]81.7%–87.5%], the BRCA2 promoter methylation in PBCs was 17.1% in COSA patients by comparison to control patients (15.4%) with p=0.0046. So, an android distribution of fat tissue revealed a higher methylation rate of the BRCA2 promoter region. Conversely, relationship between clinicopathological characteristics of tumors in subclasses and BRCA2 promoter methylation in PBCs of breast cancer patients demonstrated no statistical significant difference for any of the analyzed clinical parameters (Table 2). Table 2. Relationship Between Clinicopathological Characteristics of Tumors and BRCA2 Promoter Methylation in PBCs of Breast Cancer Patients Sensibility and specificity of the blood-based assay to distinguish breast cancer cases from controls, using BRCA2 methylation, were calculated. Reliability of the methylation of a gene such as BRCA2 to serve as a sensitive and specific blood-based breast cancer test was assessed with significant different methylation patterns between cancerous and normal PBCs. To evaluate the applicability of BRCA2 methylation in circulating DNA from PBCs as a marker for breast cancer, receiver operating characteristic (ROC) curve analysis was used (Fig. 2). The best cut-off point (16.21% of methylation) and the corresponding sensitivity (52.3%), specificity (55.8%), area under curve (AUC=0.530) and confidence interval were calculated (Table 3). The positive predictive value (PPV) was 51.4% and negative predictive value (NPV) was 56.9% in case of a cut-off point at 16.21% for the methylation. The Chi2 test (12.68) between the risk and the disease was found significant (p=0.00037). The Relative Risk (RR=1.19; 95% CI [1.08; 1.31] ) and the Odd Ratio (OR=1.39; 95% CI [1.16; 1.67]) to develop the disease by comparison with the reference fixed at 1, were calculated and were found included in their respective asymptotic 95% CI. FIG. 2. ROC curve analysis using DNA samples extracted from PBCs for discriminating between breast cancer patients and normal subjects based on methylation pattern of the BRCA2 gene. Table 3. ROC Curve Analysis of DNA Samples Extracted from PBCs Based on Methylation Proportion of the BRCA2 Gene The presenting data are promising for development of a blood-based screening method for breast cancer that relies on pathologic methylation changes, corroborating other results with different genes (Radpour et al., 2011). Further studies comparing tissue specific and blood-based methylation markers might provide valuable information as prognostic and predictive markers for breast cancer, as well as for developing novel targeted therapeutic strategies. Remy Bosviel, Julie Durif, Jiaoli Guo, Mourad Mebrek, Fabrice Kwiatkowski, Yves-Jean Bignon, and Dominique J. Bernard-Gallon

Recurrence and metastasis result in a poor prognosis for breast cancer patients. Recent studies have demonstrated that microRNAs (miRNAs) play vital roles in the development and metastasis of breast cancer. In this study, we investigated the therapeutic potential of miR-34a in breast cancer. We found that miR-34a is downregulated in breast cancer cell lines and tissues, compared with normal cell lines and the adjacent nontumor tissues, respectively. To explore the therapeutic potential of miR-34a, we designed a targeted miR-34a expression plasmid (T-VISA-miR-34a) using the T-VISA system, and evaluated its antitumor effects, efficacy, mechanism of action, and systemic toxicity. T-VISA-miR-34a induced robust, persistent expression of miR-34a, and dramatically suppressed breast cancer cell growth, migration, and invasion in vitro by downregulating the protein expression levels of the miR-34a target genes E2F3, CD44, and SIRT1. In an orthotopic mouse model of breast cancer, intravenous injection of T-VISA-miR-34a: liposomal complex nanoparticles significantly inhibited tumor growth, prolonged survival, and did not induce systemic toxicity. In conclusion, T-VISA-miR-34a lead to robust, specific overexpression of miR-34a in breast cancer cells and induced potent antitumor effects in vitro and in vivo. T-VISA-miR-34a may provide a potentially useful, specific, and safe-targeted therapeutic approach for breast cancer.

MicroRNA-34a(miR-34a), a pivotal member of the p53 network, was found to be down-regulated in multiple types of tumors and further reported as a tumor suppressor microRNA. However, the profile and biological effects of miR-34a in breast cancer are still unclear. In this study, we aimed to determine the effect of miR-34a on the growth of breast cancer and to investigate whether its effect is achieved by targeting Bcl-2 and SIRT1. We examined miR-34a levels in breast cancer cell lines and breast cancer specimens by qRT-PCR. Proliferation assay, apoptosis assay, and morphological monitoring were performed to assess the tumor suppression effect of miR-34a in breast cancer cell lines. Western blotting was used to identify the targets of miR-34a. We also investigated the anti-tumor effects of the treatment combining miR-34a with 5-FU in breast cancer cells. We found that miR-34a expression was down-regulated in 5 breast cancer cell lines compared with the immortalized normal mammary epithelial cell line 184A1, and was also down-regulated by almost 50 % in breast cancer samples compared with their corresponding adjacent non-malignant breast tissues. Ectopic restoration of miR-34a in breast cancer cells suppressed cells proliferation, invasion, and induced apoptosis. Bcl-2 and SIRT1 as the targets of miR-34a were found to be in reverse correlation with ectopic expression of miR-34a. Furthermore, the treatment combining miR-34a with 5-FU significantly showed more efficient anti-tumor effects than single treatment of miR-34a or 5-FU. Since miR-34a functions as tumor suppressor microRNA in breast cancer, modulating miR-34a level in breast cancer was suggested to be a new and useful approach of breast cancer therapy.

Background Previous studies have shown that Herpes Simplex Virus thymidine kinase (HSV-tk)/ganciclovir (GCV) comprised the most commonly used suicide gene therapy for prostate cancer, with modest results being obtained. However, novel suicide genes, such as Escherichia coli purine nucleoside phosphorylase (PNP), have been utilized to demonstrate more potent tumor killing and an enhanced bystander effect on local, non-expressing cells compared to HSV-tk.Methods PNP/fludarabine (Fludara(R); fludarabine phosphate; Berlex Labs, Richmond, CA, USA) was deliveried by prostate-specific, rat probasin-based promoter, ARR2PB. After infection of various cell lines with ADV.ARR(2)PB-PNP and administration of androgen analog, R1881, expression of PNP mRNA was detected; in vivo, the antitumor effect of the ARR(2)PB-PNP/Fludara system was monitored and analyzed, as well as animal survival.Results After in vitro infection with ADV.ARR(2)PB-PNP (multiplicity of infection = 10), LNCaP cells were more sensitive to a lower concentration Fludara (LD50, approximately 0.1 mg/ml) in the presence of R1881. Furthermore, robust bystander effects after R1881/Fludara treatment were observed in LNCaP cells after infection with bicistronic vector ADV.ARR(2)PB/PNP-IRES-EGFP in contrast to a much weaker effect in cells treated with ADV.CMV-HSV-tk/GCV. In vivo, tumor size in the ADV.ARR(2)PB-PNP/Fludara treatment group was dramatically smaller than in the control groups, and the mice treated with our system had a significantly prolonged survival, with three of eight mice surviving up to the 160-day termination point, as well as no systemic toxicity.Conclusions The ARR(2)PB-PNP/Fludara system induced massive tumor cell death and a prolonged life span without systemic cytotoxicity; therefore, it might be a more attractive strategy for suicide gene therapy of prostate cancer. Copyright (C) 2011 John Wiley & Sons, Ltd.