DIRAS3 regulates autophagy in an endometriosis epithelial cell line.

RESEARCH QUESTION: What is the role of DIRAS3 in endometriosis pathogenesis? DESIGN: Prospective patient cohort study combined with experiments in the 12Z human endometriosis epithelial cell line model to determine the role of DIRAS3 in endometriosis. Endometrium and endometriosis lesion samples were collected from premenopausal women from 24 control and 40 endometriosis patients by laparoscopic surgery. The role of DIRAS3 in endometriosis was assessed by siRNA knockdown in 12Z cells followed by proliferation, apoptosis, invasion and autophagy assays. Autophagy was induced by serum starvation and the levels of autophagy determined by assessing changes in the expression levels and localization of autophagy marker proteins, such as LC3. RESULTS: DIRAS3 mRNA showed a large increase in expression in ectopic endometriosis lesions compared with endometrium from control patients, with expression largely localized to the epithelium. DIRAS3 knockdown in 12Z endometriosis epithelial cells caused a significant reduction in the number of proliferating cells (1.6-fold, adjusted P = 0.0007) and increased apoptosis (AnnexinV/7AAD double-positive cells +48%, P = 0.01), indicating an effect on cell proliferation. Induction of autophagy by serum starvation caused significant upregulation in DIRAS3 expression after 24 h (mRNA +2.4-fold [adjusted P = 0.017], protein +8.1-fold (adjusted P = 0.029), reduced LC3I/LC3II ratio (-2.2-fold, adjusted P = 0.044) and an increase in the number of double positive LC3/DIRAS3 puncta (+2.3-fold, P = 0.02). Knockdown of DIRAS3 in serum-starved cells led to a reduction in autophagy, indicated by an overall decrease in LC3 expression and significant increase in LC3I/LC3II ratio. CONCLUSIONS: DIRAS3 is highly upregulated in endometriosis lesions. Studies in an endometriosis epithelial cell line indicate that DIRAS3 facilitates cell survival in this context by inducing autophagy.

MLLT11 Regulates Endometrial Stroma Cell Adhesion, Proliferation and Survival in Ectopic Lesions of Women with Advanced Endometriosis.

MLLT11 is a gene implicated in cell differentiation and the development and progression of human cancers, but whose role in the pathogenesis of endometriosis is still unknown. Using quantitative RT-PCR and immunohistochemistry, we analyzed 37 women with and 33 women without endometriosis for differences in MLLT11 expression. We found that MLLT11 is reduced in the ectopic stroma cells of women with advanced stage endometriosis compared to women without endometriosis. MLLT11 knockdown in control stroma cells resulted in the downregulation of their proliferation accompanied by G1 cell arrest and an increase in the expression of p21 and p27. Furthermore, the knockdown of MLLT11 was associated with increased apoptosis resistance to camptothecin associated with changes in BCL2/BAX signaling. Finally, MLLT11 siRNA knockdown in the control primary stroma cells led to an increase in cell adhesion associated with the transcriptional activation of ACTA2 and TGFB2. We found that the cellular phenotype of MLLT11 knockdown cells resembled the phenotype of the primary endometriosis stroma cells of the lesion, where the levels of MLLT11 are significantly reduced compared to the eutopic stroma cells of women without the disease. Overall, our results indicate that MLLT11 may be a new clinically relevant player in the pathogenesis of endometriosis.

The Role of Long Non-Coding RNAs in Endometriosis.

Endometriosis is a chronic gynecological disorder affecting the quality of life and fertility of many women around the world. Heterogeneous and non-specific symptoms may lead to a delay in diagnosis, with treatment options limited to surgery and hormonal therapy. Hence, there is a need to better understand the pathogenesis of the disease to improve diagnosis and treatment. Long non-coding RNAs (lncRNAs) have been increasingly shown to be involved in gene regulation but remain relatively under investigated in endometriosis. Mutational and transcriptomic studies have implicated lncRNAs in the pathogenesis of endometriosis. Single-nucleotide polymorphisms (SNPs) in lncRNAs or their regulatory regions have been associated with endometriosis. Genome-wide transcriptomic studies have identified lncRNAs that show deregulated expression in endometriosis, some of which have been subjected to further experiments, which support a role in endometriosis. Mechanistic studies indicate that lncRNAs may regulate genes involved in endometriosis by acting as a molecular sponge for miRNAs, by directly targeting regulatory elements via interactions with chromatin or transcription factors or by affecting signaling pathways. Future studies should concentrate on determining the role of uncharacterized lncRNAs revealed by endometriosis transcriptome studies and the relevance of lncRNAs implicated in the disease by in vitro and animal model studies.

LINC01133 Inhibits Invasion and Promotes Proliferation in an Endometriosis Epithelial Cell Line.

Endometriosis is a common gynecological disorder characterized by ectopic growth of endometrium outside the uterus and is associated with chronic pain and infertility. We investigated the role of the long intergenic noncoding RNA 01133 (LINC01133) in endometriosis, an lncRNA that has been implicated in several types of cancer. We found that LINC01133 is upregulated in ectopic endometriotic lesions. As expression appeared higher in the epithelial endometrial layer, we performed a siRNA knockdown of LINC01133 in an endometriosis epithelial cell line. Phenotypic assays indicated that LINC01133 may promote proliferation and suppress cellular migration, and affect the cytoskeleton and morphology of the cells. Gene ontology analysis of differentially expressed genes indicated that cell proliferation and migration pathways were affected in line with the observed phenotype. We validated upregulation of p21 and downregulation of Cyclin A at the protein level, which together with the quantification of the DNA content using fluorescence-activated cell sorting (FACS) analysis indicated that the observed effects on cellular proliferation may be due to changes in cell cycle. Further, we found testis-specific protein kinase 1 (TESK1) kinase upregulation corresponding with phosphorylation and inactivation of actin severing protein Cofilin, which could explain changes in the cytoskeleton and cellular migration. These results indicate that endometriosis is associated with LINC01133 upregulation, which may affect pathogenesis via the cellular proliferation and migration pathways.

Investigating selected adhesion molecules as urinary biomarkers for diagnosing endometriosis.

RESEARCH QUESTION: Are selected cell adhesion molecules useful as urinary biomarkers for diagnosing endometriosis? DESIGN: Prospective, longitudinal study (the Endometriosis Marker Austria) in patients who underwent laparoscopic surgery for benign gynaecological pathologies. A total of 149 patients not receiving hormonal treatment for at least 3 months prior to recruitment were included and preoperative urine protein levels of soluble vascular adhesion molecule-1 (sVCAM-1), soluble intracellular adhesion molecule-1 (sICAM-1), E-selectin and P-selectin were measured using a magnetic bead-based multiplex assay, normalized to creatinine levels of each sample. Levels were correlated with endometriosis status, menstrual cycle phase, body mass index, cigarette smoking and severity and entity of the lesions. RESULTS: Urine levels of sVCAM-1, sICAM-1, E-selectin and P-selectin did not differ between women with (n = 84) and without (n = 65) endometriosis and among subgroups. Accordingly, receiver operating characteristic analysis to examine the value of using sVCAM-1, sICAM-1, E-selectin and P-selectin levels and sVCAM/sICAM ratio to diagnose endometriosis were not significant. Whether the serum sVCAM-1 levels correlated with the urine levels of the protein in the same women was also investigated, which revealed no significant correlations for sVCAM or sICAM. CONCLUSION: Although a previous study had suggested that serum sVCAM is a promising biomarker for diagnosing endometriosis, no significant differences were found in urine levels of sVCAM-1, sICAM-1, E-selectin and P-selectin between women with and without endometriosis. Other markers should be studied in an effort to establish a truly non-invasive urinary test for diagnosing endometriosis.

Enhanced expression of TACE contributes to elevated levels of sVCAM-1 in endometriosis.

STUDY QUESTION: Are increased sVCAM-1 and sICAM-1 levels associated with tumor necrosis factor-alpha-converting enzyme (TACE) activity in endometriosis? SUMMARY ANSWER: Here we provide the first functional evidence that induced TACE activity in human endometriotic epithelial cells is at least in part responsible for the enhanced release of sVCAM-1 from these cells. WHAT IS KNOWN ALREADY: We and others have shown that serum-soluble (s)VCAM-1 levels are significantly higher in women with endometriosis, compared to disease-free controls. Experimental evidence exists suggesting a role of sICAM-1 and sVCAM-1 in the pathogenesis of endometriosis. TACE was identified as the protease responsible for phorbol 12-myristate 13-acetate (PMA)-induced VCAM-1 release in murine endothelial cells. Additionally, it has recently been shown that TACE is upregulated in the endometrial luminal epithelium of the mid-secretory phase in infertile women. STUDY DESIGN, SIZE, DURATION: This study was conducted at the Tertiary Endometriosis Referral Center of the Medical University of Vienna. Samples from a total number of 97 women were collected between July 2013 and September 2014. PARTICIPANTS/MATERIALS, SETTING, METHODS: After complete surgical exploration of the abdominopelvic cavity, 49 women with histologically proven endometriosis and 48 endometriosis-free control women were enrolled. Each participating woman contributed only one sample of eutopic endometrium and normal peritoneum, and some of the women with endometriosis contributed samples of diverse types of endometriotic lesions (in total 52 ectopic samples). Among the 49 women with endometriosis, 36 matched samples of endometriotic lesions and corresponding eutopic endometrium were collected. In order to detect sVCAM-1 and TACE protein by ELISA, peritoneal fluid (PF) samples were collected from 44 cases and 32 controls during surgery. Expression of TACE mRNA was analyzed by qRT-PCR in 111 endometrium tissue samples (28 eutopic control samples, 33 eutopic samples from women with endometriosis, 50 ectopic samples from lesions) and 37 healthy peritoneum samples. Immunohistochemistry was performed in 123 tissue samples (39 eutopic control samples, 42 eutopic samples from women with endometriosis, 42 ectopic samples from lesions) and the relation between tissue TACE protein levels and sVCAM-1 secretion was examined. PMA-induced sVCAM-1 release, and TACE- and VCAM-1-transcripts or proteins were measured in an immortalized endometriotic epithelial cell line (11Z) pre-incubated either with TACE inhibitors or following TACE siRNA knockdown. MAIN RESULTS AND THE ROLE OF CHANCE: Here, we demonstrate that TACE protein is overexpressed in epithelium of tissue samples of both eutopic endometrium and ectopic lesions of women with endometriosis compared to disease-free controls (P < 0.001 both) and that the overexpression of the protein in the lesions is due to activation of TACE gene transcription (P < 0.001). Moreover, epithelial TACE protein was significantly higher in ectopic samples than in corresponding eutopic tissue of women with the disease (P < 0.001). High endometrial tissue TACE protein expression correlated with higher serum sVCAM-1 levels (P < 0.05) but not with sICAM-1 levels. Inhibition of TACE either by TACE inhibitors or by TACE siRNA knockdown resulted in decreased PMA-induced shedding of sVCAM-1 in vitro (P < 0.005 or P < 0.01, respectively), but the TACE inhibitors did not affect transcription of TACE or VCAM-1. Additionally, we observed an upregulation of TACE in proliferative endometrial epithelium of infertile (P < 0.005), compared to fertile women. TACE was increased in infertile women with endometriosis (P = 0.051) but not in infertile women without endometriosis. LIMITATIONS, REASONS FOR CAUTION: Albeit well characterized, our control population included women with other gynecologic diseases, which may have impacted the levels of sVCAM-1 and tissue TACE expression levels, e.g. benign ovarian cysts or uterine fibroids. Thus, the results of our analysis have to be interpreted carefully and in the context of the current experimental settings. WIDER IMPLICATIONS OF THE FINDINGS: The dysregulation of TACE substrate shedding represents a promising yet relatively unexplored area of endometriosis progression and could serve as a basis for the development of new treatments of the disease. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by the Ingrid Flick Foundation. The authors have no competing interests to declare.

Enhanced expression of the stemness-related factors OCT4, SOX15 and TWIST1 in ectopic endometrium of endometriosis patients.

BACKGROUND: Current evidence suggests that endometrial-derived stem cells, spilled in the peritoneal cavity via retrograde menstruation, are key players in the establishment of endometriotic lesions. The aim of this study was to determine the presence and distribution of the stemness-related factors OCT4, SOX15, TWIST1 and DCAMLK1 in women with and without endometriosis. METHODS: Immunohistochemical analysis was used to determine stromal and epithelial expression of OCT4, SOX15, TWIST1 and DCAMLK1 in endometriosis patient (EP) endometrium (n = 69) and endometriotic tissue (n = 90) and in control endometrium (n = 50). Quantitative Real-Time PCR of OCT4, SOX15 TWIST1 and DCAMLK1 was performed in paired samples of EP endometrium and endometriotic tissue. Co-immunofluorescence staining was performed for OCT4 and SOX15. For statistical analyses we used unpaired t-test, Fisher combination test and Spearman test. For paired analyses, paired t-test and McNemar test were used. RESULTS: We detected a significant correlation between the expression of the established stem cell marker OCT4 and the stemness-related markers SOX15 (p < 0.001) and TWIST1 (p = 0.002) but not DCAMLK1. We showed a colocalization of SOX15 and OCT4 in epithelial and stromal cells of endometriotic tissue by coimmunofluorescence. A concordant expression of OCT4 and SOX15 in the same sample was observed in epithelial cells of the endometriotic tissue (71.7%). The expression of stemness-related factors was not associated with proliferative or secretory phase of the menstrual cycle in endometriosis patients but was found to be differentially expressed during the menstrual cycle in the control group. Increased expression of epithelial OCT4, SOX15 and TWIST1 was detected in endometriotic tissue compared to EP endometrium in paired (p = 0.021, p < 0.001 and p < 0.001) and unpaired analysis (p = 0.040, p < 0.001 and p = 0.001). CONCLUSION: Our findings support the hypothesis that upregulation of stem cell-related factors contribute to the establishment of endometriotic lesions. TRIAL REGISTRATION: The study was approved by the institutional review board (545/2010 on 6th of May 2014) of the Medical University of Vienna ( http://ethikkommission.meduniwien.ac.at/fileadmin/ethik/media/dokumente/register/alle_2010.pdf ).

Enhanced epithelial to mesenchymal transition (EMT) and upregulated MYC in ectopic lesions contribute independently to endometriosis.

BACKGROUND: Epithelial to mesenchymal transition (EMT) is a process in which epithelial cells lose polarity and cell-to-cell contacts and acquire the migratory and invasive abilities of mesenchymal cells. These abilities are thought to be prerequisites for the establishment of endometriotic lesions. A hallmark of EMT is the functional loss of E-cadherin (CDH1) expression in epithelial cells. TWIST1, a transcription factor that represses E-cadherin transcription, is among the EMT inducers. SNAIL, a zinc-finger transcription factor, and its close relative SLUG have similar properties to TWIST1 and are thus also EMT inducers. MYC, which is upregulated by estrogens in the uterus by an estrogen response cis-acting element (ERE) in its promoter, is associated with proliferation in endometriosis. The role of EMT and proliferation in the pathogenesis of endometriosis was evaluated by analyzing TWIST1, CDH1 and MYC expression. METHODS: CDH1, TWIST1, SNAIL and SLUG mRNA expression was analyzed by qRT-PCR from 47 controls and 74 patients with endometriosis. Approximately 42 ectopic and 62 eutopic endometrial tissues, of which 30 were matched samples, were collected during the same surgical procedure. We evaluated TWIST1 and MYC protein expression by immunohistochemistry (IHC) in the epithelial and stromal tissue of 69 eutopic and 90 ectopic endometrium samples, of which 49 matched samples were analyzed from the same patient. Concordant expression of TWIST1/SNAIL/SLUG and CDH1 but also of TWIST1 and MYC was analyzed. RESULTS: We found that TWIST1, SNAIL and SLUG are overexpressed (p < 0.001, p = 0.016 and p < 0.001) in endometriosis, while CDH1 expression was concordantly reduced in these samples (p < 0.001). Similar to TWIST1, the epithelial expression of MYC was also significantly enhanced in ectopic endometrium compared to eutopic tissues (p = 0.008). We found exclusive expression of either TWIST1 or MYC in the same samples (p = 0.003). CONCLUSIONS: Epithelial TWIST1 is overexpressed in endometriosis and may contribute to the formation of endometriotic lesions by inducing epithelial to mesenchymal transition, as CDH1 was reduced in ectopic lesions. We found exclusive expression of either TWIST1 or MYC in the same samples, indicating that EMT and proliferation contribute independently of each other to the formation of endometriotic lesions.

Extravillous trophoblast-associated ADAM12 exerts pro-invasive properties, including induction of integrin beta 1-mediated cellular spreading.

ADAM12, consisting of a membrane-bound (ADAM12L) and a secreted (ADAM12S) form, is expressed exclusively in regenerating and developing tissue as well as in certain cancer types. Strong ADAM12 expression levels have been noticed in the human placenta, and deregulated ADAM12S levels were associated with various pregnancy-related disorders including pre-eclampsia and intrauterine growth restriction. However, the role of ADAM12 in trophoblast motility has not been investigated so far. Hence, the present study aimed to investigate the specific function of the protease by using different primary trophoblast cell models. Immunofluorescence and Western blot analyses of first trimester placental tissue and differentiating primary first trimester cytotrophoblasts (CTBs) indicated strong upregulation of both of the ADAM12 isoforms during extravillous trophoblast differentiation. Functional assays involving short interfering RNA (siRNA)-mediated knockdown studies in primary CTBs and first trimester explant cultures revealed a significant repression of trophoblast motility upon partial loss of ADAM12. Conversely, isoform-specific overexpression in the ADAM12-negative trophoblast cell line SGHPL-5 enhanced the invasive capacity of these cells. We further confirmed proteolytic activity of trophoblast-derived ADAM12S by demonstrating its potential to degrade insulin-like growth factor-binding protein 3. Finally, we suggest that ADAM12S exerts its pro-migratory function in trophoblasts by inducing integrin beta 1-mediated cellular spreading.

The rs2046210 Polymorphism Is Associated with Endometriosis Risk and Elevated Estrogen Receptor 1 Expression in the Eutopic Endometrium of Women with the Disease.

In this focused genetic case-control study, we analyzed two functional single-nucleotide variants (SNVs) associated with breast cancer risk (rs2046210, rs9383590) and one risk SNV for an implantation defect and infertility (rs9340799) for their association with endometriosis susceptibility, progression and ESR1 gene regulation in endometriosis patients. The rs2046210, rs9383590 and rs9340799 SNVs were genotyped in 153 endometriosis patients and 87 control subjects with Caucasian ancestry. We analyzed the association of all SNVs with endometriosis susceptibility in all patients and in subgroups and assessed the concordance between the SNVs. Quantitative reverse transcription PCR was used to determine ESR1 gene expression in the eutopic endometrial tissue of the controls and endometriosis patients. The heterozygous rs2046210 GA genotype was associated with significantly increased endometriosis risk, particularly in younger, leaner and infertile women and with an increased ESR1 gene expression in the eutopic endometrium of these patients, compared to controls. The minor AA genotype of rs2046210 was identified as a potential risk factor for endometriosis progression in women with mild endometriosis. The results from this analysis indicate that rs2046210 may be a functional genetic variant associated with endometriosis development and progression.

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.

Hsa-mir-135a Shows Potential as A Putative Diagnostic Biomarker in Saliva and Plasma for Endometriosis.

Endometriosis is a chronic disease characterized by the implantation and proliferation of endometrial tissue outside of the uterine cavity. The nonspecific nature of the symptoms and the lack of sensitive, noninvasive diagnostic methods often lead to a significant delay in diagnosis, highlighting the need for diagnostic biomarkers. The correlation of circulating miRNAs with altered inflammatory signals seen in patients with endometriosis has raised the possibility that miRNAs can serve as biomarkers for the disease. In our study, we analyzed miRNA expression in saliva of women with and without endometriosis using a FireFly custom multiplex circulating miRNA assay. This focused panel included 28 human miRNAs, 25 of which have been previously found to be differentially expressed either in plasma, serum, and/or blood of women with endometriosis, compared to controls. We found that hsa-mir-135a was expressed significantly higher in the saliva of women with endometriosis, independent of disease stage and menstrual cycle phase. We confirmed that hsa-mir-135a also showed significantly elevated expression in the plasma of endometriosis patients. This indicates that hsa-mir-135a is a putative noninvasive biomarker of endometriosis in both saliva and plasma, but further validation studies are required to assess its clinical value as a biomarker.

Increased serum levels of mBDNF in women with minimal and mild endometriosis have no predictive power for the disease.

The objective of our pilot clinical, prospective study was to determine the serum levels of mature brain-derived neurotrophic factor, in of women with endometriosis and controls and explore whether mature brain-derived neurotrophic factor is a potential biomarker for the disease. The patients were selected from the Endometriosis Marker Austria prospective cohort study conducted at the tertiary referral certified Endometriosis Center of the Medical University of Vienna. All women underwent laparoscopic surgery because there was a suspicion of endometriosis, or the women had pelvic pain, adnexal cysts, unexplained infertility, or uterine fibroids. Our main outcome parameter was total levels of mature brain-derived neurotrophic factor in serum, measured using ELISA. Our results show that serum levels of mature brain-derived neurotrophic factor are significantly higher in women with endometriosis compared to women without endometriosis. The mean serum protein levels are significantly higher in women with rAFS stage I and II endometriosis, whereas no difference was found in women with stage III and IV endometriosis and controls. Postoperative follow-up at 6-10 weeks revealed that surgical intervention leads to equilibration of the levels of secreted mature brain-derived neurotrophic factor between women with and without endometriosis. The difference between serum mature brain-derived neurotrophic factor levels of women with endometriosis compared to women without endometriosis is independent of menstrual cycle phase and overall self-reported pelvic pain. ROC-curve analysis showed that, the mature brain-derived neurotrophic factor is not a useful biomarker for endometriosis. In conclusion, although women with stage I and II endometriosis have increased levels of mature brain-derived neurotrophic factor in serum compared to controls, the difference is not predictive for the disease. Impact statement Endometriosis is a disease that can have a significant impact on the quality of life of affected women. The gold standard for diagnosis to this day remains visualization through laparoscopic surgery with histological verification. Current studies are attempting to find a biomarker with high sensitivity and specificity, which would bypass the surgery-associated risks and would significantly reduce costs. In an attempt to elucidate whether mature serum BDNF can serve as diagnostic marker for the disease, we compared the levels of the protein in women with endometriosis to endometriosis-free controls. While our results showed that serum concentrations of the mature protein were significantly higher in women with endometriosis, we did not find this marker to have the sensitivity or specificity needed in order to allow a reliable diagnosis.

Inhibition of the mevalonate pathway affects epigenetic regulation in cancer cells.

The mevalonate pathway provides metabolites for post-translational modifications such as farnesylation, which are critical for the activity of RAS downstream signaling. Subsequently occurring regulatory processes can induce an aberrant stimulation of DNA methyltransferase (DNMT1) as well as changes in histone deacetylases (HDACs) and microRNAs in many cancer cell lines. Inhibitors of the mevalonate pathway are increasingly recognized as anticancer drugs. Extensive evidence indicates an intense cross-talk between signaling pathways, which affect growth, differentiation, and apoptosis either directly or indirectly via epigenetic mechanisms. Herein, we show data obtained by novel transcriptomic and corresponding methylomic or proteomic analyses from cell lines treated with pharmacologic doses of respective inhibitors (i.e., simvastatin, ibandronate). Metabolic pathways and their epigenetic consequences appear to be affected by a changed concentration of NADPH. Moreover, since the mevalonate metabolism is part of a signaling network, including vitamin D metabolism or fatty acid synthesis, the epigenetic activity of associated pathways is also presented. This emphasizes the far-reaching epigenetic impact of metabolic therapies on cancer cells and provides some explanation for clinical observations, which indicate the anticancer activity of statins and bisphosphonates.

Wnt-dependent T-cell factor-4 controls human etravillous trophoblast motility.

Formation of migratory extravillous trophoblasts (EVTs) is critical for human placentation and hence embryonic development. However, key regulatory growth factors, hormones, and nuclear proteins controlling the particular differentiation process remain poorly understood. Here, the role of the Wingless (Wnt)-dependent transcription factor T-cell factor-4 (TCF-4) in proliferation and motility was investigated using different trophoblast cell models. Immunofluorescence of first-trimester placental tissues revealed induction of TCF-4 and nuclear recruitment of its coactivator ß-catenin in nonproliferating EVTs, whereas membrane-associated ß-catenin decreased upon differentiation. In addition, EVTs expressed the TCF-4/ß-catenin coactivator Pygopus 2 as well as repressors of the Groucho/transducin-like enhancer of split family. Western blotting revealed Pygopus 2 expression and up-regulation of integrin α1 and nuclear TCF-4 in purified first-trimester cytotrophoblasts (CTBs) differentiating on fibronectin. Concomitantly, elevated TCF-4 mRNA, quantitated by real-time PCR, and increased TCF-dependent luciferase reporter activity were noticed in EVTs of villous explant cultures and differentiated primary CTBs. Gene silencing using specific small interfering RNA decreased TCF-4 transcript and protein levels, TCF-dependent reporter activity as well as basal and Wnt3a-stimulated migration of trophoblastic SGHPL-5 cells and primary CTBs through fibronectin-coated transwells. In contrast, proliferation of SGHPL-5 cells and primary cells, measured by cumulative cell numbers and 5-bromo-2'-deoxy-uridine labeling, respectively, was not affected. Moreover, siRNA-mediated down-regulation of TCF-4 in primary CTBs diminished markers of the differentiated EVT, such as integrin α1 and α5, Snail1, and Notch2. In summary, the data suggest that Wnt/TCF-4-dependent signaling could play a role in EVT differentiation promoting motility and expression of promigratory genes.

The Pro allele of the p53 codon 72 polymorphism is associated with decreased intratumoral expression of BAX and p21, and increased breast cancer risk.

BACKGROUND: The TP53 Arg72Pro polymorphism encodes two p53 variants with different biochemical properties. Here we investigated the impact of this polymorphism on the expression of key p53 target genes in a panel of human breast carcinomas, breast cancer risk, and age at onset. METHODOLOGY/PRINCIPAL FINDINGS: The Arg72Pro polymorphism was genotyped in 270 breast cancer patients and 221 control subjects. In addition, the Arg72Pro genotype of 116 breast tumors was determined, and correlated with intratumoral mRNA expression of TP53 and its key target genes MDM2, p21, BAX, and PERP, as quantified by qRT-PCR. We found a significantly increased breast cancer risk associated with the Pro-allele (per-allele odds ratio, 1.46; 95% confidence interval, 1.08-1.99), and a significantly later mean age at breast cancer onset for Pro/Pro patients (63.2±18 years) compared to Arg/Arg patients (58.2±15 years). The frequency of somatic TP53 inactivation was 25.4% in Arg/Arg, 20.9% in Arg/Pro, and 16.7% in Pro/Pro patients, which may reflect a higher selective pressure to mutate the Arg-allele. The median mRNA levels of p21 and BAX in the tumors of Pro-allele carriers were significantly reduced to 55.7% and 76.9% compared to Arg/Arg patients, whereas p53, MDM2 and PERP expression were hardly altered. CONCLUSIONS/SIGNIFICANCE: The p53(72Arg) variant appears to be a more potent in vivo transcription factor and tumor suppressor in human breast cancer than the p53(72Pro) variant. The Arg72Pro genotype has no significant effects in patients with TP53 mutated tumors, in which p53 is non-functional.