TGF-ß2-induced EMT is dampened by inhibition of autophagy and TNF-α treatment.

Hepatocellular carcinoma (HCC) typically develops in a chronic inflammatory setting causal to release of a plethora of growth factors and cytokines. However, the molecular effect of these cytokines on HCC progression is poorly understood. In this study, we exposed HCC cells to TGF-ß2 (Transforming Growth Factor-ß2), which resulted in a significant elevation of EMT (Epithelial to Mesenchymal Transition) like features. Molecular analysis of EMT markers showed an increase at both RNA and protein levels upon TGF-ß2 administration along with up-regulation of TGF-ß-induced Smad signaling. Induction of EMT was associated with a simultaneous increase in reactive oxygen species (ROS) and cytostasis of TGF-ß2-treated cells. Importantly, quenching of ROS resulted in a significant promotion of TGF-ß2-induced EMT. Furthermore, cells treated with TGF-ß2 also showed an enhanced autophagic flux. Interestingly, inhibition of autophagy by chloroquine-di-phosphate (CQDP) or siRNA-mediated ablation of ATG5 drastically inhibited TGF-ß2-induced EMT. Autophagy inhibition significantly increased ROS levels promoting apoptosis. It was further observed that pro-inflammatory cytokine like, TNF-α (Tumor Necrosis Factor-α) can antagonize TGF-ß2-induced response by down-regulating autophagy, increasing ROS levels and thus inhibiting EMT in HCC cells. This inhibitory effect of TNF-α is serum-independent. Transcriptomic analysis through RNA sequencing was further performed which validated that TGF-ß2-induced autophagic genes are inhibited by TNF-α treatment suppressing EMT. Our study suggests that autophagy plays a pro-metastatic role facilitating EMT by regulating ROS levels in HCC cells and TNF-α can suppress EMT by inhibiting autophagy. We provide unique mechanistic insights into the role of TGF-ß2 in HCC cells, along with appropriate cues to effectively control the disease.

MiRNomics Reveals Breast Cancer Cells Cultured on 3D Scaffolds Better Mimic Tumors in Vivo than Conventional 2D Culture.

Tissue-engineering-based three-dimensional (3D) models offer several advantages over conventional two-dimensional (2D) cultures and can mimic tissues in vivo. Although studies have analyzed the changes in the expression of genes and proteins that might mediate in vivo-like signaling, the changes in the post-transcriptional control of gene expression that are critical in fine-tuning of signaling events has never been studied. In this study, we used next-generation sequencing (NGS) to analyze the changes in the post-transcriptional regulation in MDA-MB-231 breast cancer cells cultured on 3D scaffolds. The changes in the expression of several known microRNAs were similar to the changes reported in highly invasive cancers and their profiles highly correlated with xenotumors and human breast tumors. To elucidate the role of miRNAs in modulating metastatic potential, we integrated the miRNA and the mRNA microarray data and developed networks for major pathways implicated in metastasis. From these networks, we identified several key miRNA-mRNA interactions that might contribute to the invasive behavior and aid in developing a miRNA signature for highly invasive breast cancers. This report on the differential regulation of miRNAs in breast cancer cells cultured on scaffolds demonstrates that 3D culture better mimics the tissue in vivo with novel insights into the roles of miRNAs in modulating metastatic progression.

The influence of menstrual cycle and endometriosis on endometrial methylome.

BACKGROUND: Alterations in endometrial DNA methylation profile have been proposed as one potential mechanism initiating the development of endometriosis. However, the normal endometrial methylome is influenced by the cyclic hormonal changes, and the menstrual cycle phase-dependent epigenetic signature should be considered when studying endometrial disorders. So far, no studies have been performed to evaluate the menstrual cycle influences and endometriosis-specific endometrial methylation pattern at the same time. RESULTS: Infinium HumanMethylation 450K BeadChip arrays were used to explore DNA methylation profiles of endometrial tissues from various menstrual cycle phases from 31 patients with endometriosis and 24 healthy women. The DNA methylation profile of patients and controls was highly similar and only 28 differentially methylated regions (DMRs) between patients and controls were found. However, the overall magnitude of the methylation differences between patients and controls was rather small (Δß ranging from -0.01 to -0.16 and from 0.01 to 0.08, respectively, for hypo- and hypermethylated CpGs). Unsupervised hierarchical clustering of the methylation data divided endometrial samples based on the menstrual cycle phase rather than diseased/non-diseased status. Further analysis revealed a number of menstrual cycle phase-specific epigenetic changes with largest changes occurring during the late-secretory and menstrual phases when substantial rearrangements of endometrial tissue take place. Comparison of cycle phase- and endometriosis-specific methylation profile changes revealed that 13 out of 28 endometriosis-specific DMRs were present in both datasets. CONCLUSIONS: The results of our study accentuate the importance of considering normal cyclic epigenetic changes in studies investigating endometrium-related disease-specific methylation patterns.

DNA methylome profiling of human tissues identifies global and tissue-specific methylation patterns.

BACKGROUND: DNA epigenetic modifications, such as methylation, are important regulators of tissue differentiation, contributing to processes of both development and cancer. Profiling the tissue-specific DNA methylome patterns will provide novel insights into normal and pathogenic mechanisms, as well as help in future epigenetic therapies. In this study, 17 somatic tissues from four autopsied humans were subjected to functional genome analysis using the Illumina Infinium HumanMethylation450 BeadChip, covering 486 428 CpG sites. RESULTS: Only 2% of the CpGs analyzed are hypermethylated in all 17 tissue specimens; these permanently methylated CpG sites are located predominantly in gene-body regions. In contrast, 15% of the CpGs are hypomethylated in all specimens and are primarily located in regions proximal to transcription start sites. A vast number of tissue-specific differentially methylated regions are identified and considered likely mediators of tissue-specific gene regulatory mechanisms since the hypomethylated regions are closely related to known functions of the corresponding tissue. Finally, a clear inverse correlation is observed between promoter methylation within CpG islands and gene expression data obtained from publicly available databases. CONCLUSIONS: This genome-wide methylation profiling study identified tissue-specific differentially methylated regions in 17 human somatic tissues. Many of the genes corresponding to these differentially methylated regions contribute to tissue-specific functions. Future studies may use these data as a reference to identify markers of perturbed differentiation and disease-related pathogenic mechanisms.