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.

BackgroundEpithelial 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.MethodsCDH1, 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.ResultsWe 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).ConclusionsEpithelial 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12958-015-0063-7) contains supplementary material, which is available to authorized users.

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 β-catenin in nonproliferating EVTs, whereas membrane-associated β-catenin decreased upon differentiation. In addition, EVTs expressed the TCF-4/β-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.

BackgroundThe 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 FindingsThe 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/SignificanceThe p5372Arg variant appears to be a more potent in vivo transcription factor and tumor suppressor in human breast cancer than the p5372Pro variant. The Arg72Pro genotype has no significant effects in patients with TP53 mutated tumors, in which p53 is non-functional.