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

Tight correlation between expression of the Forkhead transcription factor FOXM1 and HER2 in human breast cancer

BACKGROUND: FOXM1 regulates expression of cell cycle related genes that are essential for progression into DNA replication and mitosis. Consistent with its role in proliferation, elevated expression of FOXM1 has been reported in a variety of human tumour entities. FOXM1 is a gene of interest because recently chemical inhibitors of FOXM1 were described to limit proliferation and induce apoptosis in cancer cells in vitro, indicating that FOXM1 inhibitors could represent useful anticancer therapeutics. METHODS: Using immunohistochemistry (IHC) we systematically analysed FOXM1 expression in human invasive breast carcinomas (n = 204) and normal breast tissues (n = 46) on a tissue microarray. Additionally, using semiquantitative realtime PCR, a collection of paraffin embedded normal (n = 12) and cancerous (n = 25) breast tissue specimens as well as benign (n = 3) and malignant mammary cell lines (n = 8) were investigated for FOXM1 expression. SPSS version 14.0 was used for statistical analysis. RESULTS: FOXM1 was found to be overexpressed in breast cancer in comparison to normal breast tissue both on the RNA and protein level (e.g. 8.7 fold as measured by realtime PCR). We found a significant correlation between FOXM1 expression and the HER2 status determined by HER2 immunohistochemistry (P < 0.05). Univariate survival analysis showed a tendency between FOXM1 protein expression and unfavourable prognosis (P = 0.110). CONCLUSION: FOXM1 may represent a novel breast tumour marker with prognostic significance that could be included into multi-marker panels for breast cancer. Interestingly, we found a positive correlation between FOXM1 expression and HER2 status, pointing to a potential role of FOXM1 as a new drug target in HER2 resistant breast tumour, as FOXM1 inhibitors for cancer treatment were described recently. Further studies are underway to analyse the potential interaction between FOXM1 and HER2, especially whether FOXM1 directly activates the HER2 promoter

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

Wnt signalling in human breast cancer: expression of the putative Wnt inhibitor Dickkopf-3 (DKK3) is frequently suppressed by promoter hypermethylation in mammary tumours

INTRODUCTION: Expression of the putative Wnt signalling inhibitor Dickkopf-3 (DKK3) is frequently lost in human cancer tissues because of aberrant 5'-cytosine methylation within the DKK3 gene promoter. Since other Wnt signalling inhibitors have been reported to be targets of epigenetic inactivation in human breast cancer, we questioned if DKK3 expression is also epigenetically silenced during breast carcinogenesis and therefore might contribute to oncogenic Wnt signalling commonly found in this disease. METHODS: DKK3 mRNA expression and DKK3 promoter methylation were determined by RT-PCR, realtime PCR and methylation-specific PCR in breast cell lines (n = 9), normal breast tissues (n = 19) and primary breast carcinomas (n = 150), respectively. In vitro DNA demethylation was performed by incubating breast cell lines with 5-aza-2'-deoxycytidine and trichostatin A. DKK3 protein expression was analysed by immunohistochemistry in breast carcinomas (n = 16) and normal breast tissues (n = 8). Methylation data were statistically correlated with clinical patient characteristics. All statistical evaluations were performed with SPSS 14.0 software. RESULTS: DKK3 mRNA was downregulated in 71% (five of seven) of breast cancer cell lines and in 68% of primary breast carcinomas (27 of 40) compared with benign cell lines and normal breast tissues, respectively. A DNA demethylating treatment of breast cell lines resulted in strong induction of DKK3 mRNA expression. In tumourous breast tissues, DKK3 mRNA downregulation was significantly associated with DKK3 promoter methylation (p < 0.001). Of the breast carcinomas, 61% (92 of 150) revealed a methylated DKK3 promoter, whereas 39% (58 of 150) retained an unmethylated promoter. Loss of DKK3 expression in association with DKK3 promoter methylation (p = 0.001) was also confirmed at the protein level (p < 0.001). In bivariate analysis, DKK3 promoter methylation was not associated with investigated clinicopathological parameters except patient age (p = 0.007). CONCLUSIONS: DKK3 mRNA expression and consequently DKK3 protein expression become frequently downregulated during human breast cancer development due to aberrant methylation of the DKK3 promoter. Since DKK3 is thought to negatively regulate oncogenic Wnt signalling, DKK3 may be a potential tumour suppressor gene in normal breast tissue

Frequent expression loss of Inter-alpha-trypsin inhibitor heavy chain (ITIH) genes in multiple human solid tumors: A systematic expression analysis

<p>Abstract</p> <p>Background</p> <p>The inter-alpha-trypsin inhibitors (ITI) are a family of plasma protease inhibitors, assembled from a light chain – bikunin, encoded by <it>AMBP </it>– and five homologous heavy chains (encoded by <it>ITIH1</it>, <it>ITIH2</it>, <it>ITIH3</it>, <it>ITIH4</it>, and <it>ITIH5</it>), contributing to extracellular matrix stability by covalent linkage to hyaluronan. So far, ITIH molecules have been shown to play a particularly important role in inflammation and carcinogenesis.</p> <p>Methods</p> <p>We systematically investigated differential gene expression of the <it>ITIH </it>gene family, as well as <it>AMBP </it>and the interacting partner <it>TNFAIP6 </it>in 13 different human tumor entities (of breast, endometrium, ovary, cervix, stomach, small intestine, colon, rectum, lung, thyroid, prostate, kidney, and pancreas) using cDNA dot blot analysis (Cancer Profiling Array, CPA), semiquantitative RT-PCR and immunohistochemistry.</p> <p>Results</p> <p>We found that <it>ITIH </it>genes are clearly downregulated in multiple human solid tumors, including breast, colon and lung cancer. Thus, <it>ITIH </it>genes may represent a family of putative tumor suppressor genes that should be analyzed in greater detail in the future. For an initial detailed analysis we chose <it>ITIH2 </it>expression in human breast cancer. Loss of <it>ITIH2 </it>expression in 70% of cases (n = 50, CPA) could be confirmed by real-time PCR in an additional set of breast cancers (n = 36). Next we studied ITIH2 expression on the protein level by analyzing a comprehensive tissue micro array including 185 invasive breast cancer specimens. We found a strong correlation (p < 0.001) between ITIH2 expression and estrogen receptor (ER) expression indicating that ER may be involved in the regulation of this ECM molecule.</p> <p>Conclusion</p> <p>Altogether, this is the first systematic analysis on the differential expression of <it>ITIH </it>genes in human cancer, showing frequent downregulation that may be associated with initiation and/or progression of these malignancies.</p

New Chalcone Derivative: Synthesis, Characterization, Computational Studies and Antioxidant Activity

Ozdemir, Namik/0000-0003-3371-9874WOS: 000505999500006A new chalcone derivative, called as 1-(4-(benzylideneamino)phenyl)-3-(furan-2-yl)prop-2-en-1-one (I), was synthezised and characterized by spectral methods (infrared (IR) and proton and carbon-13 nuclear magnetic resonance (H-1- and C-13-NMR) spectroscopy). A computational study was performed by the density functional theory (DFT) method. Spectral data of compound I optimized by using 6-311G(d,p) and 6-311++G(d,p) basis sets were obtained by 6-311++G(d,p) basis set. The E-Z isomerism for newly synthesized chalcone derivative was investigated by considering four isomeric form, E/E, E/Z, Z/E and Z/Z. The results show that, as assumed and thus named, the chalcone DOI: derivative is in the E/E form. In addition, quantum chemical parameters were calculated by using DFT method with 6-311++(d,p) basis set. Antioxidant activity of compound I was determined by the feiric reducing ability of plasma (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay methods. Compound I has low antioxidant activity.Karadeniz Technical UniversityKaradeniz Teknik UniversityThis study was supported by grants from Karadeniz Technical University