Association of the Telomerase Reverse Transcriptase rs10069690 Polymorphism with the Risk, Age at Onset and Prognosis of Triple Negative Breast Cancer.

Telomerase reverse transcriptase (TERT) plays a key role in the maintenance of telomere DNA length. The rs10069690 single nucleotide variant, located in intron 4 of TERT, was found to be associated with telomere length and the risk of estrogen receptor-negative but not-positive breast cancer. This study aimed at analysis of the association of rs10069690 genotype and TERT expression with the risk, age at onset, prognosis, and clinically and molecularly relevant subtypes of breast cancer. Accordingly, rs10069690 was genotyped in a hospital-based case-control study of 403 female breast cancer patients and 246 female controls of a Central European (Austrian) study population, and the mRNA levels of TERT were quantified in 106 primary breast tumors using qRT-PCR. We found that in triple-negative breast cancer patients, the minor rs10069690 TT genotype tended to be associated with an increased breast cancer risk (OR, 1.87; 95% CI, 0.75-4.71; p = 0.155) and was significantly associated with 11.7 years younger age at breast cancer onset (p = 0.0002), whereas the CC genotype was associated with a poor brain metastasis-free survival (p = 0.009). Overall, our data show that the rs10069690 CC genotype and a high TERT expression tended to be associated with each other and with a poor prognosis. Our findings indicate a key role of rs10069690 in triple-negative breast cancer.

Discrimination between 34 of 36 Possible Combinations of Three C>T SNP Genotypes in the MGMT Promoter by High Resolution Melting Analysis Coupled with Pyrosequencing Using A Single Primer Set.

Due to its cost-efficiency, high resolution melting (HRM) analysis plays an important role in genotyping of candidate single nucleotide polymorphisms (SNPs). Studies indicate that HRM analysis is not only suitable for genotyping individual SNPs, but also allows genotyping of multiple SNPs in one and the same amplicon, although with limited discrimination power. By targeting the three C>T SNPs rs527559815, rs547832288, and rs16906252, located in the promoter of the O6-methylguanine-DNA methyltransferase (MGMT) gene within a distance of 45 bp, we investigated whether the discrimination power can be increased by coupling HRM analysis with pyrosequencing (PSQ). After optimizing polymerase chain reaction (PCR) conditions, PCR products subjected to HRM analysis could directly be used for PSQ. By analyzing oligodeoxynucleotide controls, representing the 36 theoretically possible variant combinations for diploid human cells (8 triple-homozygous, 12 double-homozygous, 12 double-heterozygous and 4 triple-heterozygous combinations), 34 out of the 36 variant combinations could be genotyped unambiguously by combined analysis of HRM and PSQ data, compared to 22 variant combinations by HRM analysis and 16 variant combinations by PSQ. Our approach was successfully applied to genotype stable cell lines of different origin, primary human tumor cell lines from glioma patients, and breast tissue samples.

Type 1 Diabetes Mellitus and the First Trimester Placenta: Hyperglycemia-Induced Effects on Trophoblast Proliferation, Cell Cycle Regulators, and Invasion.

Type 1 diabetes mellitus (T1DM) is associated with reduced fetal growth in early pregnancy, but a contributing role of the placenta has remained elusive. Thus, we investigated whether T1DM alters placental development in the first trimester. Using a protein array, the level of 60 cell-cycle-related proteins was determined in human first trimester placental tissue (gestational week 5-11) from control (n = 11) and T1DM pregnancies (n = 12). Primary trophoblasts (gestational week 7-12, n = 32) were incubated in the absence (control) or presence of hyperglycemia (25 mM D-glucose) and hyperosmolarity (5.5 mM D-glucose + 19.5 mM D-mannitol). We quantified the number of viable and dead trophoblasts (CASY Counter) and assessed cell cycle distribution (FACS) and trophoblast invasion using a transwell assay. T1DM was associated with a significant (p < 0.05) downregulation of Ki67 (-26%), chk1 (-25%), and p73 (-26%). The number of viable trophoblasts was reduced under hyperglycemia (-23%) and hyperosmolarity (-18%), whereas trophoblast invasion was increased only under hyperglycemia (+6%). Trophoblast cell death and cell cycle distribution remained unaffected. Collectively, our data demonstrate that hyperglycemia decreases trophoblast proliferation as a potential contributing factor to the reduced placental growth in T1DM in vivo.

Association of the MDM2 SNP285 and SNP309 Genetic Variants with the Risk, Age at Onset and Prognosis of Breast Cancer in Central European Women: A Hospital-Based Case-Control Study.

SNP309T>G (rs2279744) and SNP285G>C (rs117039649) in the MDM2 promoter are thought to have opposite effects on the binding of transcription factor SP1 (specificity protein 1), and consequently on MDM2 expression, p53 levels, cancer risk, age at onset, and prognosis. Here, we genotyped SNP309 and SNP285 in 406 Austrian breast cancer patients and 254 female controls. The SNP309GG genotype was associated with an increased breast cancer risk in p53 negative (OR, 1.82; 95% CI, 1.09⁻3.03; p = 0.02), but not p53 positive or unselected patients. In contrast, the SNP309TT genotype was associated with an earlier age at onset (TT, 57.0 ± 12.9; TG, 58.6 ± 13.9; GG, 59.7 ± 15.0 years; p = 0.048). 31% of SNP309TT, 26% of TG, and 13% of GG tumors were p53 positive (p = 0.034), indicating a lower selective pressure to mutate TP53 in the presence of the G-allele. Moreover, SNP309TT patients exhibited a shortened metastasis-free survival in multivariable analysis. Censoring carriers of the SNP285C-allele hardly altered the strength of these associations of SNP309, thus challenging the proposed antagonistic function of SNP285C towards SNP309G. The minor SNP285C-allele tended to be non-significantly associated with an increased breast cancer risk and a poor disease-free and metastasis-free survival, which may be bystander effects of its complete linkage disequilibrium with SNP309G. We conclude that the SNP309G-allele attenuates the p53-response and leads to a higher breast cancer risk, but also to a later onset of breast cancer and a trend towards a good prognosis.

Association of the Estrogen Receptor 1 Polymorphisms rs2046210 and rs9383590 with the Risk, Age at Onset and Prognosis of Breast Cancer.

Estrogen receptor α (ERα), encoded by the ESR1 gene, is a key prognostic and predictive biomarker firmly established in routine diagnostics and as a therapeutic target of breast cancer, and it has a central function in breast cancer biology. Genetic variants at 6q25.1, containing the ESR1 gene, were found to be associated with breast cancer susceptibility. The rs2046210 and rs9383590 single nucleotide variants (SNVs) are located in the same putative enhancer region upstream of ESR1 and were separately identified as candidate causal variants responsible for these associations. Here, both SNVs were genotyped in a hospital-based case-control study of 409 female breast cancer patients and 422 female controls of a Central European (Austrian) study population. We analyzed the association of both SNVs with the risk, age at onset, clinically and molecularly relevant characteristics and prognosis of breast cancer. We also assessed the concordances between both SNVs and the associations of each SNV conditional on the other SNV. The minor alleles of both SNVs were found to be non-significantly associated with an increased breast cancer risk. Significant associations were found in specific subpopulations, particularly in patients with an age younger than 55 years. The minor homozygotes of rs2046210 and the minor homozygotes plus heterozygotes of rs9383590 exhibited a several-years-younger age at onset than the common homozygotes, which was more pronounced in ER-positive and luminal patients. Importantly, the observed associations of each SNV were not consistently nullified upon correction for the other SNV nor upon analyses in common homozygotes for the other SNV. We conclude that both SNVs remain independent candidate causal variants.

Aberrant DNA Methylation, Expression, and Occurrence of Transcript Variants of the ABC Transporter ABCA7 in Breast Cancer.

The ABC transporter ABCA7 has been found to be aberrantly expressed in a variety of cancer types, including breast cancer. We searched for specific epigenetic and genetic alterations and alternative splicing variants of ABCA7 in breast cancer and investigated whether these alterations are associated with ABCA7 expression. By analyzing tumor tissues from breast cancer patients, we found CpGs at the exon 5-intron 5 boundary aberrantly methylated in a molecular subtype-specific manner. The detection of altered DNA methylation in tumor-adjacent tissues suggests epigenetic field cancerization. In breast cancer cell lines, DNA methylation levels of CpGs in promoter-exon 1, intron 1, and at the exon 5-intron 5 boundary were not correlated with ABCA7 mRNA levels. By qPCR involving intron-specific and intron-flanking primers, we identified intron-containing ABCA7 mRNA transcripts. The occurrence of intron-containing transcripts was neither molecular subtype-specific nor directly correlated with DNA methylation at the respective exon-intron boundaries. Treatment of breast cancer cell lines MCF-7, BT-474, SK-BR3, and MDA-MB-231 with doxorubicin or paclitaxel for 72 h resulted in altered ABCA7 intron levels. Shotgun proteomics revealed that an increase in intron-containing transcripts was associated with significant dysregulation of splicing factors linked to alternative splicing.

Differential response of arterial and venous endothelial cells to extracellular matrix is modulated by oxygen.

Binding of endothelial cell (EC) integrins to extracellular-matrix (ECM) components is one of the key events to trigger intracellular signaling that will ultimately result in proper vascular development. Even within one tissue, the endothelial phenotype differs between arteries and veins. Here, we tested the hypothesis that anchorage dependent processes, such as proliferation, viability, survival and actin organization of venous (VEC) and arterial EC (AEC) differently depend on ECM proteins. Moreover,because of different oxygen tension in AEC and VEC, we tested oxygen as a co-modulator of ECM effects. Primary human placental VEC and AEC were grown in collagens I and IV, fibronectin, laminin, gelatin and uncoated plates and exposed to 12 and 21% oxygen. Our main findings revealed that VEC are more sensitive than AEC to changes in the ECM composition. Proliferation and survival of VEC, in contrast to AEC, were profoundly increased by the presence of collagen I and fibronectin when compared with gelatin or uncoated plates. These effects were reversed by inhibition of focal adhesion kinase (Fak) and modulated by oxygen. VEC were more susceptible to the oxygen dependent ECM effects than AEC. However, no differential ECM effect on actin organization was observed between the two cell types. These data provide first evidence that AEC and VEC from the same vascular loop respond differently to ECM and oxygen in a Fak-dependent manner.

The 40bp Indel Polymorphism rs150550023 in the MDM2 Promoter is Associated with Intriguing Shifts in Gene Expression in the p53-MDM2 Regulatory Hub.

Most low-penetrance genetic risk factors for cancer are located in noncoding regions, presumably altering the regulation of neighboring genes. The poorly characterized Indel polymorphism rs150550023 (rs3730485; del1518) in the promoter of MDM2 (human homolog of mouse double minute 2) is a biologically plausible candidate genetic risk factor, which might influence the expression of MDM2, a key negative regulator of the central tumor suppressor p53. Here, we genotyped rs150550023 in a Central European hospital-based case-control study of 407 breast cancer patients and 254 female controls. mRNA levels of MDM2, p53, and the p53 target genes p21, BAX, and PERP were quantified with qRT-PCR, and p53 protein was assessed with immune histochemistry in ≈100 primary breast tumors with ascertained rs150550023 genotype. We found no evidence for an association of rs150550023 with the risk, age at onset, or prognosis of breast cancer. A possible synergism was observed with SNP309 in promoter P2 of MDM2. Mean mRNA levels of MDM2, p53, p21, and BAX were ≈1.5-3 fold elevated in TP53 wildtype tumors with the minor homozygous Del/Del genotype. However, systematic shifts in p53 protein levels or mutation rates were not observed, suggesting that the elevated p53 mRNA levels are due to regulatory feedback loops that compensate for the effects of rs150550023 on MDM2 expression.

Expression of the neonatal Fc-receptor in placental-fetal endothelium and in cells of the placental immune system.

INTRODUCTION: Starting from the second trimester of pregnancy, passive immunity is provided to the human fetus by transplacental transfer of maternal IgG. IgG transfer depends on the neonatal Fc receptor, FcRn. While FcRn localization in the placental syncytiotrophoblast (STB) has been demonstrated unequivocally, FcRn expression in placental-fetal endothelial cells (pFECs), which are part of the materno-fetal barrier, is still unclear. Therefore, this study aimed to elucidate the spatio-specific expression pattern of FcRn in placental tissue. METHODS: FcRn expression was investigated by western blotting in term placentas and in isolated human placental arterial and venous endothelial cells (HPAEC, HPVEC) using a validated affinity-purified polyclonal anti-peptide antibody against the cytoplasmic tail of FcRn α-chain. In situ localization of FcRn and IgG was studied by immunofluorescence microscopy on tissue sections of healthy term placentas. RESULTS: FcRn expression was demonstrated in placental vasculature particularly, in HPAEC, and HPVEC. FcRn was localized in cytokeratin 7+ STB and in CD31+ pFECs in terminal as well as stem villi in situ. Additionally, CD68+ placental macrophages exhibited FcRn expression in situ. Endogenous IgG partially co-localized with FcRn in STB, pFECs, and in placental macrophages. DISCUSSION: Placental FcRn expression in endothelial cells and macrophages is analogous to the expression pattern in other organs. FcRn expression in pFECs suggests an involvement of FcRn in IgG transcytosis and/or participation in recycling/salvaging of maternal IgG present in the fetal circulation. FcRn expression in placental macrophages may account for recycling of monomeric IgG and/or processing and presentation of immune complexes.

Elevated Aromatase (CYP19A1) Expression Is Associated with a Poor Survival of Patients with Estrogen Receptor Positive Breast Cancer.

Genetic variants in CYP19A1, the gene encoding aromatase, have been reported to be associated with circulating estrogen concentrations, a key risk factor for breast cancer. The mechanism underlying this association is still unclear; it has been suggested that some of these variants may alter the expression and/or activity of aromatase. Here we analyzed the expression of intra-tumoral CYP19A1 messenger RNA (mRNA) and the genotypes of rs10046, a well-characterized single nucleotide polymorphism in CYP19A1, in 138 breast cancer patients and 15 breast cancer cell lines. The genotype TT was detected in 36 patients and six cell lines, genotype CT in 55 patients and five cell lines, and genotype CC in 28 patients and four cell lines. We found no evidence for a significant association of CYP19A1 levels with rs10046 genotypes, although expression tended to be higher in tumors and cell lines with the homozygous risk genotype TT. We also found no evidence for a significant association of rs10046 genotypes with breast cancer prognosis. In contrast, high CYP19A1 expression was highly significantly associated with a poor overall, disease-free, and metastasis-free survival in estrogen receptor-positive but not negative breast cancer patients. Moreover, CYP19A1 mRNA was significantly elevated in postmenopausal patients and in patients older than 50 years, and a trend towards a positive correlation with ER status and ESR1 mRNA expression was observed. These findings highlight the key role of aromatase in estrogen receptor-positive breast cancer biology.

Hyperinsulinemia stimulates angiogenesis of human fetoplacental endothelial cells: a possible role of insulin in placental hypervascularization in diabetes mellitus.

CONTEXT: The insulin/IGF system regulates fetal and placental growth and development. In a pregnancy complicated by maternal diabetes, placentas are hypervascularized and fetal insulin levels are elevated. In the fetal circulation, insulin can act on the placenta through insulin receptors present on the fetoplacental endothelial cells. OBJECTIVE: We hypothesized that insulin exerts proangiogenic effects on the fetoplacental endothelial cells, thereby contributing to the placental hypervascularization in diabetes. DESIGN: The effect of insulin on angiogenesis and proliferation of human fetoplacental endothelial cells was investigated by a 2-dimensional network formation assay, staining for actin fibers, automatic cell counting, and cell cycle analysis. The signaling pathways involved were identified using antibodies against activated signaling proteins and pharmacological inhibitors. RESULTS: Insulin enhanced network formation by 23% (P < .05%) and caused actin reorganization. Insulin stimulated (P < .05) phosphorylation of insulin receptor (+320%), and insulin receptor substrate-1 (+140%), Akt (+177%), glycogen-synthase kinase-ß3 (+70%), and endothelial nitric oxide synthase (eNOS; +100%) increased nitric oxide production and activated Ras-related C3 botulinum toxin substrate 1 (Rac1). Insulin did not induce ERK1/2 phosphorylation or proliferation. Inhibition of phosphatidylinositol 3-kinase, eNOS, and Rac1 signaling abolished the effects on network formation. CONCLUSIONS: Elevated fetal insulin levels may contribute to the placental hypervascularization in diabetes via the phosphatidylinositol 3-kinase/Akt/eNOS pathway and involve Rac1. However, insulin does not stimulate proliferation and may need to cooperate with other growth factors.

Four and a half LIM protein 1C (FHL1C): a binding partner for voltage-gated potassium channel K(v1.5).

Four-and-a-half LIM domain protein 1 isoform A (FHL1A) is predominantly expressed in skeletal and cardiac muscle. Mutations in the FHL1 gene are causative for several types of hereditary myopathies including X-linked myopathy with postural muscle atrophy (XMPMA). We here studied myoblasts from XMPMA patients. We found that functional FHL1A protein is completely absent in patient myoblasts. In parallel, expression of FHL1C is either unaffected or increased. Furthermore, a decreased proliferation rate of XMPMA myoblasts compared to controls was observed but an increased number of XMPMA myoblasts was found in the G(0)/G(1) phase. Furthermore, low expression of K(v1.5), a voltage-gated potassium channel known to alter myoblast proliferation during the G(1) phase and to control repolarization of action potential, was detected. In order to substantiate a possible relation between K(v1.5) and FHL1C, a pull-down assay was performed. A physical and direct interaction of both proteins was observed in vitro. In addition, confocal microscopy revealed substantial colocalization of FHL1C and K(v1.5) within atrial cells, supporting a possible interaction between both proteins in vivo. Two-electrode voltage clamp experiments demonstrated that coexpression of K(v1.5) with FHL1C in Xenopus laevis oocytes markedly reduced K(+) currents when compared to oocytes expressing K(v1.5) only. We here present the first evidence on a biological relevance of FHL1C.