Promoter hypermethylation of the SFRP2 gene is a high-frequent alteration and tumor-specific epigenetic marker in human breast cancer

<p>Abstract</p> <p>Background</p> <p>We have previously reported that expression of the Wnt antagonist genes <it>SFRP1 </it>and <it>SFRP5 </it>is frequently silenced by promoter hypermethylation in breast cancer. SFRP2 is a further Wnt inhibitor whose expression was recently found being downregulated in various malignancies. Here we investigated whether SFRP2 is also implicated in human breast cancer, and if so whether <it>SFRP2 </it>promoter methylation might serve as a potential tumor biomarker.</p> <p>Methods</p> <p>We analyzed <it>SFRP2 </it>mRNA expression and <it>SFRP2 </it>promoter methylation in 10 breast cell lines, 199 primary breast carcinomas, 20 matched normal breast tissues and 17 cancer-unrelated normal breast tissues using RT-PCR, realtime PCR, methylation-specific PCR and Pyrosequencing, respectively. SFRP2 protein expression was assessed by immunohistochemistry on a tissue microarray. Proliferation assays after transfection with an <it>SFRP2 </it>expression vector were performed with mammary MCF10A cells. Statistical evaluations were accomplished with SPSS 14.0 software.</p> <p>Results</p> <p>Of the cancerous breast cell lines, 7/8 (88%) lacked <it>SFRP2 </it>mRNA expression due to <it>SFRP2 </it>promoter methylation (<it>P </it>< 0.001). <it>SFRP2 </it>expression was substantially restored in most breast cell lines after treatment with 5-aza-2'-deoxycytidine and trichostatin A. In primary breast carcinomas SFRP2 protein expression was strongly reduced in 93 of 125 specimens (74%). <it>SFRP2 </it>promoter methylation was detected in 165/199 primary carcinomas (83%) whereas all cancer-related and unrelated normal breast tissues were not affected by <it>SFRP2 </it>methylation. <it>SFRP2 </it>methylation was not associated with clinicopathological factors or clinical patient outcome. However, loss of SFRP2 protein expression showed a weak association with unfavorable patient overall survival (<it>P </it>= 0.071). Forced expression of <it>SFRP2 </it>in mammary MCF10A cells substantially inhibited proliferation rates (<it>P </it>= 0.045).</p> <p>Conclusion</p> <p>The <it>SFRP2 </it>gene is a high-frequent target of epigenetic inactivation in human breast cancer. Its methylation leads to abrogation of <it>SFRP2 </it>expression, conferring a growth advantage to epithelial mammary cells. This altogether supports a tumor suppressive function of <it>SFRP2</it>. Although clinical patient outcome was not associated with <it>SFRP2 </it>methylation, the high frequency of this epimutation and its putative specificity to neoplastic cells may qualify <it>SFRP2 </it>promoter methylation as a potential candidate screening marker helping to improve early breast cancer detection.</p

Nanoscale Topography and Poroelastic Properties of Model Tissue Breast Gland Basement Membranes

Basement membranes (BMs) are thin layers of condensed extracellular matrix proteins serving as permeability filters, cellular anchoring sites, and barriers against cancer cell invasion. It is believed that their biomechanical properties play a crucial role in determining cellular behavior and response, especially in mechanically active tissues like breast glands. Despite this, so far, relatively little attention has been dedicated to their analysis because of the difficulty of isolating and handling such thin layers of material. Here, we isolated BMs derived from MCF10A spheroids—three-dimensional breast gland model systems mimicking in vitro the most relevant phenotypic characteristics of human breast lobules—and characterized them by atomic force microscopy, enhanced resolution confocal microscopy, and scanning electron microscopy. By performing atomic force microscopy height-clamp experiments, we obtained force-relaxation curves that offered the first biomechanical data on isolated breast gland BMs to our knowledge. Based on enhanced resolution confocal microscopy and scanning electron microscopy imaging data, we modeled the system as a polymer network immersed in liquid and described it as a poroelastic material. Finite-element simulations matching the experimental force-relaxation curves allowed for the first quantification, to our knowledge, of the bulk and shear moduli of the membrane as well as its water permeability. These results represent a first step toward a deeper understanding of the mechanism of tensional homeostasis regulating mammary gland activity as well as its disruption during processes of membrane breaching and metastatic invasion

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Calculation of temperature distribution and effective temperature in high burnup fuel of WWER-1000

The problem of finding an effective temperature is highly important. This characteristic provides calculation of the power effect reactivity (PER), which defines safe and stable operation of nuclear reactor. The numerical experiment, which is considered on average and maximum heat load conditions, is put to find out changing of the effective temperature in the high burnup fuel of WWER-1000. In the course of the experiment mathematical statement of the problem is made, numerical solution of which is found with using the finite differences approximation of both control equations and border conditions. Also, the method of simple iteration is used for calculating temperature distributions, according to determination of the effective temperature. The linear approximation was obtained basing on effective and maximum temperatures depending on the burnup

ITIH5 mediates epigenetic reprogramming of breast cancer cells

Extracellular matrix (ECM) is known to maintain epithelial integrity. In carcinogenesis ECM degradation triggers metastasis by controlling migration and differentiation including cancer stem cell (CSC) characteristics. The ECM-modulator inter- α-trypsin inhibitor heavy chain family member five (ITIH5) was recently identified as tumor suppressor potentially involved in impairing breast cancer progression but molecular mechanisms underlying its function are still elusive

Conversations under a Tung Tree

<p>Secreted frizzled related protein 3 (SFRP3) contains a cysteine-rich domain (CRD) that shares homology with Frizzled CRD and regulates WNT signaling. Independent studies showed epigenetic silencing of <i>SFRP3</i> in melanoma and hepatocellular carcinoma. Moreover, a tumor suppressive function of SFRP3 was shown in androgen-independent prostate and gastric cancer cells. The current study is the first to investigate <i>SFRP3</i> expression and its potential clinical impact on non-small cell lung carcinoma (NSCLC). WNT signaling components present on NSCLC subtypes were preliminary elucidated by expression data of The Cancer Genome Atlas (TCGA). We identified a distinct expression signature of relevant WNT signaling components that differ between adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). Of interest, canonical WNT signaling is predominant in LUAD samples and non-canonical WNT signaling is predominant in LUSC. In line, high SFRP3 expression resulted in beneficial clinical outcome for LUAD but not for LUSC patients. Furthermore, <i>SFRP3</i> mRNA expression was significantly decreased in NSCLC tissue compared to normal lung samples. TCGA data verified the reduction of <i>SFRP3</i> in LUAD and LUSC patients. Moreover, DNA hypermethylation of <i>SFRP3</i> was evaluated in the TCGA methylation dataset resulting in epigenetic inactivation of <i>SFRP3</i> expression in LUAD, but not in LUSC, and was validated by pyrosequencing of our NSCLC tissue cohort and <i>in vitro</i> demethylation experiments. Immunohistochemistry confirmed SFRP3 protein downregulation in primary NSCLC and indicated abundant expression in normal lung tissue. Two adenocarcinoma gain-of-function models were used to analyze the functional impact of SFRP3 on cell proliferation and regulation of <i>CyclinD1</i> expression <i>in vitro</i>. Our results indicate that <i>SFRP3</i> acts as a novel putative tumor suppressor gene in adenocarcinoma of the lung possibly regulating canonical WNT signaling.</p

Promoter methylation-associated loss of ID4 expression is a marker of tumour recurrence in human breast cancer

<p>Abstract</p> <p>Background</p> <p>Inhibitor of DNA binding/Inhibitor of differentiation 4 (<it>ID4</it>) is a critical factor for cell proliferation and differentiation in normal vertebrate development. <it>ID4</it> has regulative functions for differentiation and growth of the developing brain. The role of <it>ID1</it>, <it>ID2</it> and <it>ID3</it> are expected to be oncogenic due to their overexpression in pancreatic cancer and colorectal adenocarcinomas, respectively. Aside from these findings, loss of <it>ID3</it> expression was demonstrated in ovarian cancer. The aim of the present study was to reveal the factual role of <it>ID4</it> in carcinogenesis in more detail, since its role for the pathogenesis of human breast cancer has been discussed controversially, assigning both oncogenic and tumour suppressive functions. </p> <p>Methods</p> <p><it>ID4</it> promoter methylation, <it>ID4</it> mRNA expression and <it>ID4</it> protein expression were analysed in primary human breast cancer specimens using methylation-specific PCR (MSP) (n=170), semiquantitative realtime RT-PCR (n=46) and immunhistochemistry (n=3), respectively. In order to demonstrate a functional association of <it>ID4</it> promoter methylation with its gene silencing, we performed DNA demethylation analysis with four human breast cell lines using MSP and semiquantitative realtime RT-PCR. In addition, we performed correlations of <it>ID4</it> promoter methylation with <it>ID4</it> mRNA and <it>ID4</it> protein expression in matched samples of breast tumour and corresponding normal tissue. We carried out statistical analyses in order to find correlations between <it>ID4</it> promoter methylation and clinicopathological parameters. </p> <p>Results</p> <p>Frequent <it>ID4</it> promoter methylation was observed in primary breast cancer samples (69%, 117/170). We found a tight correlation (P<0.0001) between <it>ID4</it> promoter methylation and loss of <it>ID4</it> expression in primary breast cancer 3 specimens. Demethylating treatment with breast cancer cell lines was associated with clear ID4 mRNA re-expression. Tumours with <it>ID4</it> promoter methylation showed distinct loss of <it>ID4</it> expression on both transcription and protein level. Interestingly, <it>ID4</it> promoter methylation was a factor for unfavourable recurrence-free survival (P=0.036) and increased risk for lymph node metastasis (P=0.030). </p> <p>Conclusion</p> <p>ID4 is indeed a novel tumour suppressor gene in normal human breast tissue and is epigenetically silenced during cancer development, indicating increased risk for tumour relapse. Thus, <it>ID4</it> methylation status could serve as a prognostic biomarker in human breast cancer.</p

The ubiquitin-like molecule interferon-stimulated gene 15 (ISG15) is a potential prognostic marker in human breast cancer

INTRODUCTION: ISG15 is an ubiquitin-like molecule that is strongly upregulated by type I interferons as a primary response to diverse microbial and cellular stress stimuli. However, alterations in the ISG15 signalling pathway have also been found in several human tumour entities. To the best of our knowledge, in the current study we present for the first time a systematic characterisation of ISG15 expression in human breast cancer and normal breast tissue both at the mRNA and protein level. METHOD: Using semiquantitative real-time PCR, cDNA dot-blot hybridisation and immunohistochemistry, we systematically analysed ISG15 expression in invasive breast carcinomas (n = 910) and normal breast tissues (n = 135). ISG15 protein expression was analysed in two independent cohorts on tissue microarrays; in an initial evaluation set of 179 breast carcinomas and 51 normal breast tissues; and in a second large validation set of 646 breast carcinomas and 10 normal breast tissues. In addition, a collection of benign and malignant mammary cell lines (n = 9) were investigated for ISG15 expression. RESULTS: ISG15 was overexpressed in breast carcinoma cells compared with normal breast tissue, both at the RNA and protein level. Recurrence-free (p = 0.030), event-free (p = 0.001) and overall (p = 0.001) survival analyses showed a significant correlation between ISG15 overexpression and unfavourable prognosis. CONCLUSION: Therefore, ISG15 may represent a novel breast tumour marker with prognostic significance and may be helpful in selecting patients for and predicting response to the treatment of human breast cancer

Identifizierung von Synemin (SYNM) als einen neuen DNA-Methylierungsmarker und Charakterisierung des putativen Onkogens Karyopherin Alpha 2 (KPNA2) für das humane Mammakarzinom

The understanding of tumor biological consequences of aberrantly expressed genes and the identification of novel DNA methylation markers are fundamental prerequisites for the development of future cancer therapies as well as for the improvement of prognosis and prediction assessment of breast cancer patients. This work describes the identification of a novel DNA methylation marker (Synemin) and the functional characterization of a putative oncoprotein (KPNA2) for human breast cancer. The intermediate filament Synemin (SYNM) is crucial for the linkage of the extracellular matrix and the intermediate filament network. Altered expression of intermediate filaments is an important mechanism for carcinogenesis and tumor invasion. In this study the role of SYNM for human breast cancer was investigated for the first time. Initially performed RNA expression analysis demonstrated a highly significant SYNM loss in invasive breast tumors and abundant expression in normal breast tissues. Immunohistochemical SYNM protein expression analysis revealed a myoepithelial expression pattern in normal breast tissues. SYNM protein was drastically reduced in 70% and completely lost in 56% of all investigated invasive breast cancers. Promoter analysis revealed a CpG island in a SYNM promoter sequence relevant for transcription. Promoter methylation analysis (MSP-technique) revealed a tumor specific SYNM promoter methylation in 26.7% of invasive tumors (n=195). Pyrosequencing technique revealed CpG methylation in the entire promoter sequence analyzed. We found a highly significant association between SYNM promoter methylation and SYNM expression loss in invasive breast tumors and human cancer cell lines. This association was confirmed functionally, since the in vitro demethylation of cancer cell lines by 5'-aza-2'-deoxycytidine resulted in significant SYNM re-expression. Therefore, tumor-specific SYNM promoter methylation was confirmed as a cardinal mechanism for SYNM gene silencing in breast cancer. Patients with SYNM methylation had a significantly shortened disease-free survival, poor tumor differentiation and advanced metastatic disease. SYNM methylation was identified as independent prognostic marker for a 2.9-fold increased risk of tumor recurrence. Interestingly, nodal-positive breast cancer patients without SYNM methylation had an unexpectedly good prognosis. SYNM methylation could therefore serve as a new prognostic methylation marker for breast cancer and improve the risk assessment of high-risk patients. The intermediate filament SYNM could also have a tumor suppressive effect with respect to its biological function in breast myoepithelial cells. Dysfunction of the nuclear transport machinery at the level of soluble import and export receptors (karyopherins) is associated with tumorigenesis and tumor progression. Retrospective studies already proposed the tumor-specific overexpression of the nuclear import factor karyopherin alpha 2 (KPNA2) as a new prognostic marker for human breast cancer. On this basis a detailed, functional characterization of the phenotypic effects of the differential KPNA2 expression on breast cancer cells was performed. Prerequisite for this was the establishment of KPNA2 overexpressing cell lines. An inducible Tet-On KPNA2 expression system was stably integrated into malignant MCF7 cells (MCF7/TR/KPNA2). Benign non- transformed MCF10A cells were transiently transfected with a constitutive KPNA2 expression vector (MCF10A/KPNA2). MCF7 wild type cells (WT) had a stronger KPNA2 expression (x 5.1) than MCF10A WT cells and proliferated faster (+16%). KPNA2 overexpression in MCF7/TR/KPNA2 clones further increased cell proliferation (+15%). MCF10A WT cells showed a highly significant higher cell-matrix adhesion of 36% compared to MCF7 WT cells. MCF10A/KPNA2 cells showed a drastic reduction of adhesion (-21%), which was similar to that of malignant MCF7 WT cells. In contrast, migration was significantly increased in MCF10A/KPNA2 cells (+68.9%) and MCF7/TR/KPNA2 clones (+25.5%). MCF10A/KPNA2 cells almost achieved the migration ability of malignant MCF7 WT cells. In Consequence, the demonstrated effects on proliferation, adhesion and migration led to an increased colony spreading in MCF10A/KPNA2 (+190%) and MCF7/TR/KPNA2 (+41%) tumor cells. The phenotypic effects described here may facilitate the invasion and metastatic potential of breast tumor cells. Thus, the oncogenic value of KPNA2 could be confirmed in vitro. One possible mechanism could be the KPNA2 forced nuclear translocation of RAC-1 and p65. These oncogenes are proven KPNA2 interaction partners and crucial for cell cycle control, adhesion and migration