Wenyi Qin

Circulating microRNAs in breast cancer and healthy subjects

BackgroundIt has been demonstrated that extracellular mRNA can be detected in the circulation. Our hypothesis was that circulating miRNAs are also present and differentially expressed in the serum of breast cancer patients compared to controls.FindingsWe measured miRNA in the serum of samples with and without the addition of miRNA prior to analysis. To test our RNA extraction efficiency, we spiked-in serial dilutions of single-strand C elegens miR-39 (cel-miR-39) and human miR-145 (has-miR-145) into goat serum and a 10 year old human serum specimen. We next analyzed miR-16, -145, and -155 in archived serum specimens from 21 participants, 13 of whom did and 8 of whom did not have breast cancer. We were able to detect the miRNAs from all the serum samples to which the miRNAs had been added. We were also able to detect endogenous miR-16, -145, and -155 in all serum samples. While the expression of all three miRNAs was similar in samples from healthy women compared to those with breast cancer, women with progesterone receptor (PR, p = 0.016) positive tumors had higher miR-155 expression than tumors that were negative for these receptors.Conclusion1) RNA species can be detected in archived serum; 2) miR-155 may be differentially expressed in the serum of women with hormone sensitive compared to women with hormone insensitive breast cancer. Screening serum for miRNAs that predict the presence of breast cancer is feasible, and may be useful for breast cancer detection.

Soy Isoflavones Have an Antiestrogenic Effect and Alter Mammary Promoter Hypermethylation in Healthy Premenopausal Women

We determined if soy isoflavones have dose-related estrogenic and methylation effects. Thirty-four healthy premenopausal women were randomized to 40 mg or 140 mg isoflavones daily through one menstrual cycle. Breast specific and systemic estrogenic effects were assessed measuring the estrogenic marker complement (C)3 and changes in cytology, whereas methylation assessment of 5 cancer related genes (p16, RASSF1A, RAR β 2, ER, and CCND2) was performed on intraductal specimens. Serum genistein significantly increased after consuming both isoflavone doses. Cytology did not significantly change at either isoflavone dose. Serum C3 levels posttreatment were inversely related to change in serum genistein ( r =–0.76, P = 0.0045) in women consuming low but not high dose isoflavones. The RAR β 2 hypermethylation increase posttreatment correlated with the posttreatment genistein level considering the entire group ( r = 0.67, P = 0.0017) and those receiving high-dose isoflavones ( r = 0.68, P = 0.021). At the low but not the high isoflavone dose, CCND2 hypermethylation increase correlated with posttreatment genistein levels ( r = 0.79, P = 0.011). In summary, the inverse correlation between C3 and genistein suggests an antiestrogenic effect. Isoflavones induced dose-specific changes in RAR β 2 and CCND2 gene methylation, which correlated with genistein levels. This work provides novel insights into estrogenic and methylation effects of dietary isoflavones.

Quantitative evaluation of DNA hypermethylation in malignant and benign breast tissue and fluids

The assessment of DNA had demonstrated altered methylation in malignant compared to benign breast tissue. The purpose of our study was to (i) confirm the predictive ability of methylation assessment in breast tissue, and (ii) use the genes found to be cancer predictive in tissue to evaluate the diagnostic potential of hypermethylation assessment in nipple aspirate fluid (NAF) and mammary ductoscopic (MD) samples. Quantitative methylation specific (qMS)‐PCR was conducted on three specimen sets: 44 malignant (CA) and 34 normal (NL) tissue specimens, 18 matched CA, adjacent normal (ANL) tissue and NAF specimens, and 119 MD specimens. Training and validation tissue sets were analyzed to determine the optimal group of cancer predictive genes for NAF and MD analysis. NAF and MD cytologic review were also performed. Methylation of CCND‐2, p16, RAR‐β and RASSF‐1a was significantly more prevalent in tumor than in normal tissue specimens. Receiver operating characteristic curve analysis demonstrated an area under the curve of 0.96. For the 18 matched CA, ANL and NAF specimens, the four predictive genes identified in cancer tissue contained increased methylation in CA vs. ANL tissue; NAF samples had higher methylation than ANL specimens. Methylation frequency was higher in MD specimens from breasts with cancer than benign samples for p16 and RASSF‐1a. In summary, i) routine quantitative DNA methylation assessment in NAF and MD samples is possible, and ii) genes hypermethylated in malignant breast tissue are also altered in matched NAF and in MD samples, and may be useful to assist in early breast cancer detection.

Trans-Resveratrol Alters Mammary Promoter Hypermethylation in Women at Increased Risk for Breast Cancer

Trans-resveratrol, present in high concentration in the skin of red grapes and red wine, has a dose-dependent antiproliferative effect in vitro, prevents the formation of mammary tumors, and has been touted as a chemopreventive agent. Based upon in vitro studies demonstrating that trans-resveratrol downregulates the expression of 1) DNA methyltransferases and 2) the cancer promoting prostaglandin (PG)E2, we determined if trans-resveratrol had a dose-related effect on DNA methylation and prostaglandin expression in humans. Thirty-nine adult women at increased breast cancer risk were randomized in double-blind fashion to placebo, 5 or 50 mg trans-resveratrol twice daily for 12 wk. Methylation assessment of 4 cancer-related genes (p16, RASSF-1α, APC, CCND2) was performed on mammary ductoscopy specimens. The predominant resveratrol species in serum was the glucuronide metabolite. Total trans-resveratrol and glucuronide metabolite serum levels increased after consuming both trans-resveratrol doses (P < .001 for both). RASSF-1α methylation decreased with increasing levels of serum trans-resveratrol (P = .047). The change in RASSF-1α methylation was directly related to the change in PGE2 (P = .045). This work provides novel insights into the effects of trans-resveratrol on the breast of women at increased breast cancer risk, including a decrease in methylation of the tumor suppressor gene RASSF-1α. Because of the limited sample size, our findings should be validated in a larger study.

Methylation and miRNA effects of resveratrol on mammary tumors vs. normal tissue.

We reported that resveratrol decreased DNA methyltransferase (DNMT) 1 and 3b expression in vitro and demethylates tumor suppressor RASSF-1a in women at increased breast cancer risk. We investigated the effects of resveratrol on DNMT and miRNA expression in normal and tumor mammary tissue in a rodent model of estrogen dependent mammary carcinoma. Eighty-nine female ACI rats received estradiol plus: low dose (lo) resveratrol, high dose (hi) resveratrol, 5-aza-2-deoxycytidine (Aza), a known inhibitor of DNMTs, or control (no additional treatment). After 21 wk of treatment, animals were sacrificed and mammary glands harvested. Matched tumor/normal tissues were available from 36 rats. DMNT3b (but not DNMT1) differed in tumor vs. normal tissue after lo (P = .04) and hi (P = .007) resveratrol and Aza treatment. With hi resveratrol, DNMT3b decreased in tumor but increased normal tissue. Hi resveratrol increased miR21, −129, −204, and −489 >twofold in tumor and decreased the same miRs in normal tissue 10–50% compared to control. There was an inverse association between DNMT3b and miR129, −204, and −489 in normal and/or tumor tissue. Treatment with resveratrol differentially influences tumor vs. normal tissue DNMT3b and miRNA expression. This mechanism of action of resveratrol to influence mammary carcinogenesis warrants further investigation.

High basic fibroblast growth factor levels in nipple aspirate fluid are correlated with breast cancer

PURPOSEThe angiogenic basic fibroblast growth factor (bFGF) and vascular endothelial growth factor are important in malignant breast epithelial growth. Nipple aspirate fluid (NAF) is a physiologic fluid collected noninvasively that contains proteins secreted by the breast ductal epithelium and may contain markers of breast cancer. The purpose of this study was to determine whether high concentrations of bFGF and vascular endothelial growth factor in NAF would be associated with in situ and invasive breast cancer, and whether prostate-specific antigen, a marker in NAF associated with breast cancer, would improve our ability to determine which subjects had the disease. METHODSBoth bivariate and multivariate analyses were performed to determine the effects of race, menopausal status, bFGF concentration, and prostate-specific antigen on cancer risk. Bivariate analysis was also performed to determine the relationship between vascular endothelial growth factor concentration and cancer risk. RESULTSMean NAF bFGF levels were higher in women with breast cancer than in those without (19.2 vs 1.74 ng/g). Vascular endothelial growth factor was not associated with breast cancer. Race and menopausal status did not significantly affect the relationship between bFGF and cancer risk. bFGF, race, and menopausal status were each independent predictors of breast cancer, with bFGF being the most important. With knowledge of all three variables, the model was 89.9% sensitive and 69.0% specific in predicting which women had breast cancer. Adding prostate-specific antigen increased the sensitivity to 90.9% and the specificity to 83.3%. In subjects with NAF bFGF > 150 ng/g and prostate-specific antigen < 100 ng/g, 94.1% (32/34) of subjects had cancer. For women with NAF prostate-specific antigen > 100 ng/ g and bFGF < 150 ng/g, 90.5% were cancer free. CONCLUSIONSbFGF concentration in NAF is directly associated with breast cancer, regardless of race and menopausal status. NAF bFGF may prove helpful in the early detection of breast cancer.

99mTc-peptide-peptide nucleic acid probes for imaging oncogene mRNAs in tumours.

Summary Imaging oncogene mRNA in tumours would provide a powerful tool for the early detection of occult malignant lesions. The goal was to prepare a chimera consisting of a dodecamer antisense peptide nucleic acid (PNA) specific for c‐MYC oncogene overexpressed in human breast cancer cells and a chelating moiety that facilitates quantitative radiolabelling with 99mTc and evaluate it for hybridization and tissue distribution in laboratory animals. The pentapeptide chelator‐PNA dodecamer specific for c‐MYC mRNA was extended from a solid support by 9‐fluorenylmethyloxycarbonyl (Fmoc) coupling. Similarly, a chelator‐PNA chimera with four central mismatches was also prepared which served as a control. The chimeras were purified, characterized and evaluated for hybridization to c‐MYC mRNA by fluorescent, real‐time polymerase chain reaction (RT‐PCR). The chimeras were labelled with 99mTc and their tissue distribution was examined in athymic nude mice bearing experimental human breast tumours. 99mTc radiolabelling was quantitative and presented a single peak in reversed phase liquid chromatography. Fluorescent real‐time polymerase chain reactions using primer and fluorescent probe sets previously calculated for c‐MYC mRNA demonstrated inhibition of reverse transcription by the c‐MYC specific chimera as compared to that of the control. Tissue distribution studies of antisense and mismatch chimeras at 4 h and 24 h after administration displayed modest accumulation in the liver, and appreciable levels in tumours. These observations suggest that 99mTc‐peptide‐PNA probes might be useful for imaging gene expression in tumours, and the approach is worthy of further investigation.

External imaging of CCND1, MYC, and KRAS oncogene mRNAs with tumor-targeted radionuclide-PNA-peptide chimeras.

Abstract: In 2005, breast cancer will kill approximately 40,410 women in the U.S., and pancreatic cancer will kill approximately 31,800 men and women in the U.S. Clinical examination and mammography, the currently accepted breast cancer screening methods, miss almost half of breast cancers in women younger than 40 years, approximately one‐quarter of cancers in women aged 40‐49 years, and one‐fifth of cancers in women over 50 years old. Pancreatic cancer progresses rapidly, with only 1% of patients surviving more than 5 years after diagnosis. However, if the disease is diagnosed when it is localized, the 5‐year survival is approximately 20%. It would be beneficial to detect breast cancer and pancreatic cancer at the earliest possible stage, when multimodal therapy with surgery, radiotherapy, and chemotherapy have the greatest chance of prolonging survival. Human estrogen receptor‐positive breast cancer cells typically display elevated levels of Myc protein due to overexpression of MYC mRNA, elevated cyclin D1 protein due to overexpression of CCND1 mRNA, and elevated insulin‐like growth factor 1 receptor (IGF1R) due to overexpression of IGF1R mRNA. We hypothesized that scintigraphic detection of MYC or CCND1 peptide nucleic acid (PNA) probes with an IGF1 peptide loop on the C‐terminus, and a Tc‐99m‐chelator peptide on the N‐terminus, could measure levels of MYC or CCND1 mRNA noninvasively in human IGF1R‐overexpressing MCF7 breast cancer xenografts in immunocompromised mice. Similarly, human pancreatic cancer cells typically display elevated levels of KRAS mRNA and elevated IGF1R. Hence, we also hypothesized that a KRAS Tc‐99m‐chelator PNA‐peptide probe could detect overexpression of KRAS mRNA in pancreatic cancer xenografts by scintigraphic imaging, or by positron emission tomography (PET) with a KRAS Cu‐64‐chelator PNA‐peptide. Human MCF7 breast cancer xenografts in immunocompromised mice were imaged scintigraphically 4‐24 h after tail‐vein administration of MYC or CCND1 Tc‐99m‐chelator PNA‐peptides, but not after administration of mismatch controls. Similarly, human Panc‐1 pancreatic cancer cells xenografts were imaged scintigraphically 4 and 24 h after tail‐vein administration of a KRAS Tc‐99m‐chelator PNA‐peptide, and AsPC1 xenografts were imaged by PET 4 and 24 h after tail‐vein adminstration of a KRAS Cu‐64‐chelator PNA‐peptide. The radioprobes distributed normally to the kidneys, livers, tumors, and other tissues. External molecular imaging of oncogene mRNAs in solid tumors with radiolabel‐PNA‐peptide chimeras might in the future provide additional genetic characterization of pre‐invasive and invasive breast cancers.

The 8‐epimer of prostaglandin F2α, a marker of lipid peroxidation and oxidative stress, is decreased in the nipple aspirate fluid of women with breast cancer

Breast cancer (BC), a worldwide disease with increasing incidence, develops from ductal/lobular epithelium. Nipple aspirate fluid (NAF), secreted from the breast ducts and lobules, can be analyzed to assess breast metabolic activity. Whether lipid peroxidation in the mammary gland promotes or prevents tumorigenesis is unclear. Malondialdehyde (MDA) and the 8‐epimer of Prostaglandin F2α (8‐iso‐PGF2α), two lipid peroxidation markers, were studied in milk (n = 10), NAF (n = 140) and plasma (n = 35) samples. MDA was detected in all plasma, in 80% of milk samples and in 95% of NAF samples. MDA levels in NAF and plasma were significantly higher than in milk (p = 0.016 and p = 0.029, respectively). We found no significant difference between levels of MDA in NAF samples from BC patients compared to healthy controls. 8‐iso‐PGF2α was detectable in all samples. 8‐iso‐PGF2α median levels in NAF were significantly higher than in both milk and plasma (p < 0.0001). The highest 8‐iso‐PGF2α levels were found in NAF from healthy women, significantly higher than in women with BC (p < 0.0001). No significant differences were found in both markers after the age‐adjustment. High levels of lipid peroxidation products in NAF suggest their in situ production in the nonlactating breast. Active lipid peroxidation may have a physiologic role in the normal mammary gland. Lower levels of 8‐iso‐PGF2α in NAF from BC patients suggest altered production of arachidonic acid metabolites during breast carcinogenesis.

Nipple aspirate fluids from women with breast cancer contain increased levels of group IIa secretory phospholipase A2

Arachidonic acid, a bioactive molecule metabolized into prostaglandins and leukotrienes, contributes to cellular proliferation and tumor progression. Group IIa secretory phospholipase A2 (sPLA2-IIa) can facilitate arachidonate release from cellular phospholipids, suggesting its involvement in tumor evolution. Analysis of breast nipple aspirate fluid (NAF), a noninvasive research tool, allows the identification of biomarkers of breast cancer. We sought to determine whether sPLA2-IIa expression might be related to breast cancer development or progression. sPLA2-IIa expression was evaluated in NAF samples from 110 women (57 women with and 53 without breast cancer) using ELISA and western blotting; ultrastructural immunolocalization was performed in epithelial cells floating in NAF. Immunocytochemistry revealed that sPLA2-IIa is a constitutive intracellular protein suggesting that breast ductal cells synthesize and secrete the 14xa0kDa protein in NAF. Among all 110 subjects, sPLA2-IIa expression was significantly increased both in NAF and within ductal epithelial cells from cancer containing breasts. While in healthy women menopausal status did not influence sPLA2-IIa expression (Pxa0=xa00.457), among patients with breast cancer there was a significant down-regulation in postmenopausal subjects (Pxa0<xa00.0001). Moreover, sPLA2-IIa concentration in NAF from breast cancer patients was positively correlated with tumor stage (r2xa0=xa00.979, Pxa0=xa00.0012), suggesting an active secretion/accumulation of the enzyme in NAF based on tumor burden. sPLA2-IIa activity may serve a dual role in breast carcinogenesis, beneficial in its release of arachidonate and detrimental in the metabolic conversion of arachadonic acid into prostaglandins and leukotrienes.