Decitabine-induced DNA methylation-mediated transcriptomic reprogramming in human breast cancer cell lines; the impact of DCK overexpression

Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor, is tested in combination with conventional anticancer drugs as a treatment option for various solid tumors. Although epigenome modulation provides a promising avenue in treating resistant cancer types, more studies are required to evaluate its safety and ability to normalize the aberrant transcriptional profiles. As deoxycytidine kinase (DCK)-mediated phosphorylation is a rate-limiting step in DAC metabolic activation, we hypothesized that its intracellular overexpression could potentiate DAC’s effect on cell methylome and thus increase its therapeutic efficacy. Therefore, two breast cancer cell lines, JIMT-1 and T-47D, differing in their molecular characteristics, were transfected with a DCK expression vector and exposed to low-dose DAC (approximately IC20). Although transfection resulted in a significant DCK expression increase, further enhanced by DAC exposure, no transfection-induced changes were found at the global DNA methylation level or in cell viability. In parallel, an integrative approach was applied to decipher DAC-induced, methylation-mediated, transcriptomic reprogramming. Besides large-scale hypomethylation, accompanied by up-regulation of gene expression across the entire genome, DAC also induced hypermethylation and down-regulation of numerous genes in both cell lines. Interestingly, TET1 and TET2 expression halved in JIMT-1 cells after DAC exposure, while DNMTs’ changes were not significant. The protein digestion and absorption pathway, containing numerous collagen and solute carrier genes, ranking second among membrane transport proteins, was the top enriched pathway in both cell lines when hypomethylated and up-regulated genes were considered. Moreover, the calcium signaling pathway, playing a significant role in drug resistance, was among the top enriched in JIMT-1 cells. Although low-dose DAC demonstrated its ability to normalize the expression of tumor suppressors, several oncogenes were also up-regulated, a finding, that supports previously raised concerns regarding its broad reprogramming potential. Importantly, our research provides evidence about the involvement of active demethylation in DAC-mediated transcriptional reprogramming.publishedVersio

Decitabine potentiates efficacy of doxorubicin in a preclinical trastuzumab-resistant HER2-positive breast cancer models

Acquired drug resistance and metastasis in breast cancer (BC) are coupled with epigenetic deregulation of gene expression. Epigenetic drugs, aiming to reverse these aberrant transcriptional patterns and sensitize cancer cells to other therapies, provide a new treatment strategy for drug-resistant tumors. Here we investigated the ability of DNA methyltransferase (DNMT) inhibitor decitabine (DAC) to increase the sensitivity of BC cells to anthracycline antibiotic doxorubicin (DOX). Three cell lines representing different molecular BC subtypes, JIMT-1, MDA-MB-231 and T-47D, were used to evaluate the synergy of sequential DAC + DOX treatment in vitro. The cytotoxicity, genotoxicity, apoptosis, and migration capacity were tested in 2D and 3D cultures. Moreover, genome-wide DNA methylation and transcriptomic analyses were employed to understand the differences underlying DAC responsiveness. The ability of DAC to sensitize trastuzumab-resistant HER2-positive JIMT-1 cells to DOX was examined in vivo in an orthotopic xenograft mouse model. DAC and DOX synergistic effect was identified in all tested cell lines, with JIMT-1 cells being most sensitive to DAC. Based on the whole-genome data, we assume that the aggressive behavior of JIMT-1 cells can be related to the enrichment of epithelial-to-mesenchymal transition and stemness-associated pathways in this cell line. The four-week DAC + DOX sequential administration significantly reduced the tumor growth, DNMT1 expression, and global DNA methylation in xenograft tissues. The efficacy of combination therapy was comparable to effect of pegylated liposomal DOX, used exclusively for the treatment of metastatic BC. This work demonstrates the potential of epigenetic drugs to modulate cancer cells' sensitivity to other forms of anticancer therapy.publishedVersio

Analysis of biological networks via hyperbolic network embedding

Εθνικό Μετσόβιο Πολυτεχνείο--Μεταπτυχιακή Εργασία. Διεπιστημονικό-Διατμηματικό Πρόγραμμα Μεταπτυχιακών Σπουδών (Δ.Π.Μ.Σ.) "Επιστήμη Δεδομένων και Μηχανική Μάθηση

The amyloid interactome: Exploring protein aggregation

<div><p>Protein-protein interactions are the quintessence of physiological activities, but also participate in pathological conditions. Amyloid formation, an abnormal protein-protein interaction process, is a widespread phenomenon in divergent proteins and peptides, resulting in a variety of aggregation disorders. The complexity of the mechanisms underlying amyloid formation/amyloidogenicity is a matter of great scientific interest, since their revelation will provide important insight on principles governing protein misfolding, self-assembly and aggregation. The implication of more than one protein in the progression of different aggregation disorders, together with the cited synergistic occurrence between amyloidogenic proteins, highlights the necessity for a more universal approach, during the study of these proteins. In an attempt to address this pivotal need we constructed and analyzed the human amyloid interactome, a protein-protein interaction network of amyloidogenic proteins and their experimentally verified interactors. This network assembled known interconnections between well-characterized amyloidogenic proteins and proteins related to amyloid fibril formation. The consecutive extended computational analysis revealed significant topological characteristics and unraveled the functional roles of all constituent elements. This study introduces a detailed protein map of amyloidogenicity that will aid immensely towards separate intervention strategies, specifically targeting sub-networks of significant nodes, in an attempt to design possible novel therapeutics for aggregation disorders.</p></div

The amyloid interactome.

<p>Interaction data for the creation of this network were gathered from the publicly available database IntAct [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173163#pone.0173163.ref045" target="_blank">45</a>] and Cytoscape [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173163#pone.0173163.ref051" target="_blank">51</a>] was used as a visualization tool (Interactive network available at <a href="http://83.212.109.111/amyloid_interactome" target="_blank">http://83.212.109.111/amyloid_interactome</a>). The network consists of 353 nodes and 1178 edges. Proteins are depicted as nodes and interactions as edges. Red-coloured nodes represent known <i>in vivo</i> amyloidogenic proteins, whereas yellow-coloured nodes represent <i>in vitro</i> amyloid-forming proteins or proteins related to amyloid fibril formation (see also Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173163#pone.0173163.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173163#pone.0173163.t002" target="_blank">2</a>). Green-coloured nodes are proteins, listed as other interaction partners. Hubs and bottlenecks are depicted as triangles (▲) and squares (■), respectively. Protein-nodes, which are both hubs and bottlenecks are shown as diamonds (◆). Important molecular chaperones are highlighted with a blue outline.</p

Clustering analysis of the amyloid interactome.

<p>The 11 clusters with 3 or more nodes of the amyloid interaction network, derived utilizing the MCL algorithm [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173163#pone.0173163.ref097" target="_blank">97</a>]. Cytoscape [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173163#pone.0173163.ref051" target="_blank">51</a>] was used as a visualization tool. The visual legend summarizes the shortcuts of node colour and node shape (See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173163#pone.0173163.g001" target="_blank">Fig 1</a>). The highly connected subnetwork of the first cluster within the amyloid interactome reveals the strong affinity between 7 amyloidogenic proteins (cluster 1—red nodes) and the integral representation of the proteins presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173163#pone.0173163.t002" target="_blank">Table 2</a> (cluster 1—yellow nodes) (Interactive cluster subnetworks available at <a href="http://83.212.109.111/amyloid_interactome" target="_blank">http://83.212.109.111/amyloid_interactome</a>).</p

Subnetworks of molecular chaperones participating in the amyloid interactome.

<p>3 important subnetworks were isolated from the entire amyloid interactome: (A) Subnetwork of Hsp90 co-chaperone Cdc37, Hsc70-interacting protein, Hsp 90-alpha, Hsc71 and their first neighbors, (B) Subnetwork of Serum albumin and Hsc70-interacting protein and their first neighbors and (C) Subnetwork of Clusterin, Large proline-rich protein BAG6 and their first neighbors. The aforementioned proteins, having chaperone or co-chaperone activity, were found to play a pivotal role in the integrity of the interactome (See section Network Analysis Based on Graph Theory). A highly selective and direct correlation of Serum albumin and 6 amyloidogenic proteins was observed (B), whereas indirect interactions between Serum albumin and 2 amyloidogenic proteins were recorded (A). Hsc70-interacting protein is a significant element of the interactome, since it conciliates interactions between Apolipoproteins and ACys or ATTR (A,B). Clusterin synergistically with Large proline-rich protein BAG6 interferes with APrp and Aβ2M (C). The finding that more than one chaperones mediate the interconnection between different amyloidogenic proteins deserves further investigation.</p

The dataset of 28 proteins related to in vivo amyloid fibril formation.

<p>The dataset of 28 proteins related to in vivo amyloid fibril formation.</p

The dataset of 13 proteins related to amyloid fibril formation.

<p>The dataset of 13 proteins related to amyloid fibril formation.</p

Decitabine-induced DNA methylation-mediated transcriptomic reprogramming in human breast cancer cell lines; the impact of DCK overexpression

Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor, is tested in combination with conventional anticancer drugs as a treatment option for various solid tumors. Although epigenome modulation provides a promising avenue in treating resistant cancer types, more studies are required to evaluate its safety and ability to normalize the aberrant transcriptional profiles. As deoxycytidine kinase (DCK)-mediated phosphorylation is a rate-limiting step in DAC metabolic activation, we hypothesized that its intracellular overexpression could potentiate DAC’s effect on cell methylome and thus increase its therapeutic efficacy. Therefore, two breast cancer cell lines, JIMT-1 and T-47D, differing in their molecular characteristics, were transfected with a DCK expression vector and exposed to low-dose DAC (approximately IC20). Although transfection resulted in a significant DCK expression increase, further enhanced by DAC exposure, no transfection-induced changes were found at the global DNA methylation level or in cell viability. In parallel, an integrative approach was applied to decipher DAC-induced, methylation-mediated, transcriptomic reprogramming. Besides large-scale hypomethylation, accompanied by up-regulation of gene expression across the entire genome, DAC also induced hypermethylation and down-regulation of numerous genes in both cell lines. Interestingly, TET1 and TET2 expression halved in JIMT-1 cells after DAC exposure, while DNMTs’ changes were not significant. The protein digestion and absorption pathway, containing numerous collagen and solute carrier genes, ranking second among membrane transport proteins, was the top enriched pathway in both cell lines when hypomethylated and up-regulated genes were considered. Moreover, the calcium signaling pathway, playing a significant role in drug resistance, was among the top enriched in JIMT-1 cells. Although low-dose DAC demonstrated its ability to normalize the expression of tumor suppressors, several oncogenes were also up-regulated, a finding, that supports previously raised concerns regarding its broad reprogramming potential. Importantly, our research provides evidence about the involvement of active demethylation in DAC-mediated transcriptional reprogramming