High-density array analysis of DNA methylation in Tamoxifen-resistant breast cancer cell lines

Roughly two-thirds of all breast cancers are ERα-positive and can be treated with the antiestrogen, Tamoxifen, however resistance occurs in 33% of women who take the drug for more than 5 y. Aberrant DNA methylation, an epigenetic mechanism that alters gene expression in cancer, is thought to play a role in this resistance. To develop an understanding of Tamoxifen-resistance and identify novel pathways and targets of aberrant methylation, DNA from MCF-7 breast cancer cells and Tamoxifen-resistant derivatives, TMX2–11 and TMX2–28, were analyzed using the Illumina HumanMethylation450 BeadChip. Normalizing against MCF-7 values, ERα-positive TMX2–11 had 4000 hypermethylated sites and ERα-negative TMX2–28 had over 33 000. Analysis of CpG sites altered in both TMX2–11 and TMX2–28 revealed that the Tamoxifen-resistant cell lines share 3000 hypermethylated and 200 hypomethylated CpGs. ZNF350 and MAGED1, two genes hypermethylated in both cell lines, were examined in greater detail. Treatment with 5-aza-2′deoxycitidine caused a significant reduction in promoter methylation of both ZNF350 and MAGED1 and a corresponding increase in expression in TMX2–28. A similar relationship between methylation and expression was not detected in TMX2–11. Our findings are indicative of the variable responses to methylation-targeted breast cancer therapy and highlight the need for biomarkers that accurately predict treatment outcome.     

Genome-scale analysis of DNA methylation in colorectal cancer using Infinium HumanMethylation450 BeadChips

Illumina’s Infinium HumanMethylation450 BeadChip arrays were used to examine genome-wide DNA methylation profiles in 22 sample pairs from colorectal cancer (CRC) and adjacent tissues and 19 colon tissue samples from cancer-free donors. We show that the methylation profiles of tumors and healthy tissue samples can be clearly distinguished from one another and that the main source of methylation variability is associated with disease status. We used different statistical approaches to evaluate the methylation data. In general, at the CpG-site level, we found that common CRC-specific methylation patterns consist of at least 15,667 CpG sites that were significantly different from either adjacent healthy tissue or tissue from cancer-free subjects. Of these sites, 10,342 were hypermethylated in CRC, and 5,325 were hypomethylated. Hypermethylated sites were common in the maximum number of sample pairs and were mostly located in CpG islands, where they were significantly enriched for differentially methylated regions known to be cancer-specific. In contrast, hypomethylated sites were mostly located in CpG shores and were generally sample-specific. Despite the considerable variability in methylation data, we selected a panel of 14 highly robust candidates showing methylation marks in genes SND1, ADHFE1, OPLAH, TLX2, C1orf70, ZFP64, NR5A2, and COL4A. This set was successfully cross-validated using methylation data from 209 CRC samples and 38 healthy tissue samples from The Cancer Genome Atlas consortium (AUC = 0.981 [95% CI: 0.9677–0.9939], sensitivity = 100% and specificity = 82%). In summary, this study reports a large number of loci with novel differential methylation statuses, some of which may serve as candidate markers for diagnostic purposes.     

Proteomics of xenografted human breast cancer indicates novel targets related to tamoxifen resistance

Tamoxifen is the most frequently used drug for hormone therapy of breast cancer patients, even though a high percentage of women are (or become) refractory to this treatment. The proteins involved in tamoxifen resistance of breast tumor cells as well as the mechanisms by which they interact, are still unknown. Some years ago, we established the xenograft breast tumor 3366, sensitive to tamoxifen and the 3366/TAM, resistant to tamoxifen, derived after two years of in vivo passages of the parental 3366 under tamoxifen treatment. Here, we compare the protein expression levels of both xenografts. 2-DE of proteins from total cell extracts showed very high reproducibility among tumors from each group (tamoxifen sensitive and tamoxifen resistant). The heuristic clustering analysis of these gels pooled them correctly in both groups. Twelve proteins were found up-regulated in the tamoxifen-resistant line, while nine were down-regulated. The proteins differentially expressed were identified by MS and sequence database analysis. Biological functions of these proteins are related to cell-cell adhesion and interaction, signal transduction, DNA and protein synthesis machinery, mitochondrial respiratory chain, oxidative stress processes and apoptosis. Three of the identified proteins (ALG-2 interacting protein and two GDP-dissociation inhibitors) could be directly involved in the resistance phenomenon.     

Improved reproducibility in genome-wide DNA methylation analysis for PAXgene-fixed samples compared with restored formalin-fixed and paraffin-embedded DNA

Formalin fixation has been the standard method for conservation of clinical specimens for decades. However, a major drawback is the high degradation of nucleic acids, which complicates its use in genome-wide analyses. Unbiased identification of biomarkers, however, requires genome-wide studies, precluding the use of the valuable archives of specimens with long-term follow-up data. Therefore, restoration protocols for DNA from formalin-fixed and paraffin-embedded (FFPE) samples have been developed, although they are cost-intensive and time-consuming. An alternative to FFPE and snap-freezing is the PAXgene Tissue System, developed for simultaneous preservation of morphology, proteins, and nucleic acids. In the current study, we compared the performance of DNA from either PAXgene or formalin-fixed tissues to snap-frozen material for genome-wide DNA methylation analysis using the Illumina 450K BeadChip. Quantitative DNA methylation analysis demonstrated that the methylation profile in PAXgene-fixed tissues showed, in comparison with restored FFPE samples, a higher concordance with the profile detected in frozen samples. We demonstrate, for the first time, that DNA from PAXgene conserved tissue performs better compared with restored FFPE DNA in genome-wide DNA methylation analysis. In addition, DNA from PAXgene tissue can be directly used on the array without prior restoration, rendering the analytical process significantly more time- and cost-effective.     

A ferrocenyl derivative of hydroxytamoxifen elicits an estrogen receptor-independent mechanism of action in breast cancer cell lines

The aim of this work was to investigate the mechanism of action of ferrocifen (Fc-OH-TAM), the ferrocenyl analog of 4-hydroxy-tamoxifen (OH-TAM), which is the active metabolite of tamoxifen, the drug most widely prescribed for treatment of hormone-dependent breast cancers. Fc-OH-TAM showed an anti-proliferative effect on the six breast cancer cell lines tested, 3 ERα positive (MCF-7, T-47D, ZR-75-1) and 3 ERα negative (MDA-MB-231, SKBR-3, Hs578-T) whatever their ER (estrogen receptor) status. However, the mechanism of action of the ferrocenyl derivative appeared to differ depending on the status of the ERα. Analysis of cell cycle distribution revealed that Fc-OH-TAM first recruits cells in the S phase in both ERα positive and ERα negative cells. In the presence of ERα, Fc-OH-TAM allowed cell cycle progression, with a subsequent blockade in G0/G1, whereas in the absence of ERα, cells remained in the S phase. Significant production of ROS was observed only in the presence of Fc-OH-TAM in both ERα positive and negative breast cancer cell lines. Within our experimental conditions, this ROS production is associated with cell cycle arrest and senescence rather than apoptosis. In the presence of ERα, Fc-OH-TAM seems to mainly act in the same way as OH-TAM but also induces an additional cytotoxic effect not mediated by the receptor. Our data suggest that this cytotoxic effect of Fc-OH-TAM is expressed via a mechanism of action distinct from the non-genomic pathway observed with high doses of OH-Tamoxifen.     

Prolylcarboxypeptidase regulates proliferation, autophagy, and resistance to 4-hydroxytamoxifen-induced cytotoxicity in estrogen receptor-positive breast cancer cells

Endocrine therapy with tamoxifen (TAM) significantly improves outcomes for patients with estrogen receptor-positive breast cancer. However, intrinsic (de novo) or acquired resistance to TAM occurs in a significant proportion of treated patients. To identify genes involved in resistance to TAM, we introduced full-length cDNA expression library into estrogen receptor-positive MCF7 cells and exposed them to a cytotoxic dose of 4-hydroxytamoxifen (4OHTAM). Four different library inserts were isolated from surviving clones. Re-introduction of the genes individually into naive MCF7 cells made them resistant to 4OHTAM. Cells overexpressing these genes had an increase in acidic autophagic vacuoles induced by 4OHTAM, suggesting their role in autophagy. One of them, prolylcarboxypeptidase (PRCP), was investigated further. Overexpression of PRCP increased cell proliferation, boosted several established markers of autophagy, including expression of LC3-2, sequestration of monodansylcadaverine, and proteolysis of BSA in an ER-α dependent manner, and increased resistance to 4OHTAM. Conversely, knockdown of endogenous PRCP in MCF7 cells increased cell sensitivity to 4OHTAM and at the same time decreased cell proliferation and expression of LC3-2, sequestration of monodansylcadaverine, and proteolysis of BSA. Inhibition of enzymatic activity of PRCP enhanced 4OHTAM-induced cytotoxicity in MCF7 cells. Cells with acquired resistance to 4OHTAM exhibited increased PRCP activity, although inhibition of PRCP prevented development of 4OHTAM resistance in parental MCF7 cells and restored response to 4OHTAM in MCF7 cells with acquired resistance to 4OHTAM. Thus, we have for the first time identified PRCP as a resistance factor for 4OHTAM resistance in estrogen receptor-positive breast cancer cells.     

耐三苯氧胺乳腺癌异种移植瘤动物模型建立与评价

目的：建立耐三苯氧胺（TAM）人乳腺癌的裸鼠移植瘤模型,为研究和治疗乳腺癌对TAM耐药提供研究工具。方法：采用雌激素受体阳性,对TAM耐药的人乳腺癌细胞系LCC2,接种于BALB/c裸鼠皮下,观察肿瘤生长趋势,用免疫组化方法进行鉴定。结果：在接种细胞数大于5×106/只时,Matrigel能够显著促进移植瘤的生长。肿瘤组织病理学检测证实为浸润性导管癌,且Pgp和Her-2为阳性表达。结论：该方法建立的耐TAM人乳腺癌移植瘤模型,周期短,成瘤率高,保留了与细胞系相同的肿瘤生物学特征。     

Progestin inhibition of cell death in human breast cancer cell lines

Previously, we have shown that progestins both stimulate proliferation of the progesterone receptor (PR)-rich human breast cancer cell line T47D and protect from cell death, in charcoal-stripped serum-containing medium. To lessen the variability inherent in different preparations of serum, we decided to further characterize progestin inhibition of cell death using serum starvation to kill the cells, and find that progestins protect from serum-starvation-induced apoptosis in T47D cells. This effect exhibits specificity for progestins and is inhibited by the antiprogestin RU486. While progestin inhibits cell death in a dose–responsive manner at physiological concentrations, estradiol-17β surprisingly does not inhibit cell death at any concentration from 0.001 nM to 1 μM. Progestin inhibition of cell death also occurs in at least two other human breast cancer cell lines, one with an intermediate level of PR, MCF-7 cells, and, surprisingly, one with no detectable level of PR, MDA-MB-231 cells. Further, we have found progestin inhibition of cell death caused by the breast cancer chemotherapeutic agents doxorubicin and 5-fluorouracil. These data are consistent with the building body of evidence that progestins are not the benign hormones for breast cancer they have been so long thought to be, but may be harmful both for undiagnosed cases and those undergoing treatment.     

Reducing the risk of false discovery enabling identification of biologically significant genome-wide methylation status using the HumanMethylation450 array

Background

The Illumina HumanMethylation450 BeadChip (HM450K) measures the DNA methylation of 485,512 CpGs in the human genome. The technology relies on hybridization of genomic fragments to probes on the chip. However, certain genomic factors may compromise the ability to measure methylation using the array such as single nucleotide polymorphisms (SNPs), small insertions and deletions (INDELs), repetitive DNA, and regions with reduced genomic complexity. Currently, there is no clear method or pipeline for determining which of the probes on the HM450K bead array should be retained for subsequent analysis in light of these issues.

Results

We comprehensively assessed the effects of SNPs, INDELs, repeats and bisulfite induced reduced genomic complexity by comparing HM450K bead array results with whole genome bisulfite sequencing. We determined which CpG probes provided accurate or noisy signals. From this, we derived a set of high-quality probes that provide unadulterated measurements of DNA methylation.

Conclusions

Our method significantly reduces the risk of false discoveries when using the HM450K bead array, while maximising the power of the array to detect methylation status genome-wide. Additionally, we demonstrate the utility of our method through extraction of biologically relevant epigenetic changes in prostate cancer.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-51) contains supplementary material, which is available to authorized users.     

Novel imprinted single CpG sites found by global DNA methylation analysis in human parthenogenetic induced pluripotent stem cells

Genomic imprinting is the process of epigenetic modification whereby genes are expressed in a parent-of-origin dependent manner; it plays an important role in normal growth and development. Parthenogenetic embryos contain only the maternal genome. Parthenogenetic embryonic stem cells could be useful for studying imprinted genes. In humans, mature cystic ovarian teratomas originate from parthenogenetic activation of oocytes; they are composed of highly differentiated mature tissues containing all three germ layers. To establish human parthenogenetic induced pluripotent stem cell lines (PgHiPSCs), we generated parthenogenetic fibroblasts from ovarian teratoma tissues. We compared global DNA methylation status of PgHiPSCs with that of biparental human induced pluripotent stem cells by using Illumina Infinium HumanMethylation450 BeadChip array. This analysis identified novel single imprinted CpG sites. We further tested DNA methylation patterns of two of these sites using bisulfite sequencing and described novel candidate imprinted CpG sites. These results confirm that PgHiPSCs are a powerful tool for identifying imprinted genes and investigating their roles in human development and diseases.     

Regulation of fas ligand expression in breast cancer cells by estrogen: functional differences between estradiol and tamoxifen

During neoplastic growth and metastasis, the immune system responds to the tumor by developing both cellular and humoral immune responses. In spite of this active response, tumor cells escape immune surveillance. We previously showed that FasL expression by breast tumor plays a central role in the induction of apoptosis of infiltrating Fas-immune cells providing the mechanism for tumor immune privilege. In the present study, we showed that FasL in breast tissue is functionally active, and estrogen and tamoxifen regulate its expression. We identified an estrogen recognizing element like-motif in the promoter region of the FasL gene, suggesting direct estrogen effects on FasL expression. This was confirmed by an increase in FasL expression in both RNA and protein levels in hormone sensitive breast cancer cells treated with estradiol. This effect is receptor mediated since tamoxifen blocked the estrogenic effect. Interestingly, tamoxifen also inhibited FasL expression in estrogen-depleted conditions. Moreover, an increase in FasL in breast cancer cells induces apoptosis in Fas bearing T cells and, tamoxifen blocks the induction of apoptosis. These studies provide evidence that tamoxifen inhibits FasL expression, allowing the killing of cancer cells by activated lymphocytes. This partially explains the protective effect of tamoxifen against breast cancer.     

AIB1 is required for the acquisition of epithelial growth factor receptor-mediated tamoxifen resistance in breast cancer cells

Acquired resistance to tamoxifen has become a serious obstacle in breast cancer treatment. The underlying mechanism responsible for this condition has not been completely elucidated. In this study, a tamoxifen-resistant (Tam-R) MCF-7 breast cancer cell line was developed to mimic the occurrence of acquired tamoxifen resistance as seen in clinical practice. Increased expression levels of HER1, HER2 and the estrogen receptor (ER)-AIB1 complex were found in tamoxifen-resistant cells. EGF stimulation and gefitinib inhibition experiments further demonstrated that HER1/HER2 signaling and AIB1 were involved in the proliferation of cells that had acquired Tam resistance. However, when AIB1 was silenced with AIB1-siRNA in Tam-R cells, the cell growth stimulated by the HER1/HER2 signaling pathway was significantly reduced, and the cells were again found to be inhibited by tamoxifen. These results suggest that the AIB1 protein could be a limiting factor in the HER1/HER2-mediated hormone-independent growth of Tam-R cells. Thus, AIB1 may be a new therapeutic target, and the removal of AIB1 may decrease the crosstalk between ER and the HER1/HER2 pathway, resulting in the restoration of tamoxifen sensitivity in tamoxifen-resistant cells.     

Induction of tamoxifen resistance in breast cancer cells by ELF electromagnetic fields

The incidence of breast cancer in western societies has been rising ever since the Second World War. Besides the exposure to a multitude of new chemical compounds, electromagnetic field exposure has been linked to breast cancer through a radiation-mediated anti-melatonin pathway. We investigated, whether low-frequency electromagnetic field exposure interferes with the anti-estrogenic activity of tamoxifen. Two different clones of the breast cancer cell line MCF-7 were exposed to highly homogeneous 50Hz electromagnetic fields and IC(50) values were calculated from dose-response curves of tamoxifen at various field intensities. An intensity-dependent shift of tamoxifen dose-response curves to higher concentrations with a maximal response at 1.2muT was observed. Hypothetically, electromagnetic field exposure could contribute to tamoxifen resistance observed in breast cancer after long-term treatment.