Engineered reversal of drug resistance in cancer cells - metastases suppressor factors as change agents

Building molecular correlates of drug resistance in cancer and exploiting them for therapeutic intervention remains a pressing clinical need. To identify factors that impact drug resistance herein we built a model that couples inherent cell-based response toward drugs with transcriptomes of resistant/sensitive cells. To test this model, we focused on a group of genes called Metastasis Suppressor Genes (MSGs) that influence aggressiveness and metastatic potential of cancers. Interestingly, modeling of 84 000 drug response transcriptome combinations predicted multiple MSGs to be associated with resistance of different cell types and drugs. As a case study, on inducing MSG levels in a drug resistant breast cancer line resistance to anticancer drugs caerulomycin, camptothecin and topotecan decreased by more than 50–60 %, in both culture conditions and also in tumors generated in mice, in contrast to control un-induced cells. To our knowledge, this is the first demonstration of engineered reversal of drug resistance in cancer cells based on a model that exploits inherent cellular response profiles

Promoter-proximal transcription factor binding is transcriptionally active when coupled with nucleosome repositioning in immediate vicinity

Previous studies have analyzed patterns of transcription, Transcription Factor (TF) binding or mapped nucleosome occupancy across the genome. These suggest that the three aspects are genetically connected but the cause and effect relationships are still unknown. For example, physiologic TF binding studies involve many TFs, consequently, it is difficult to assign nucleosome reorganization to the binding site occupancy of any particular TF. Therefore, several aspects remain unclear: does TF binding influence nucleosome (re)organizations locally or impact the chromatin landscape at a more global level; are all or only a fraction of TF binding a result of reorganization in nucleosome occupancy and do all TF binding and associated changes in nucleosome occupancy result in altered gene expression? With these in mind, following characterization of two states (before and after induction of a single TF of choice) we determined: (i) genomic binding sites of the TF, (ii) promoter nucleosome occupancy and (iii) transcriptome profiles. Results demonstrated that promoter-proximal TF binding influenced expression of the target gene when it was coupled to nucleosome repositioning at or close to its binding site in most cases. In contrast, only in few cases change in target gene expression was found when TF binding occurred without local nucleosome reorganization

Metastases suppressor NM23-H2 interaction with G-quadruplex DNA within c-MYC promoter nuclease hypersensitive element induces c-MYC expression

Regulatory influence of the G-quadruplex or G4 motif present within the nuclease hypersensitive element (NHE) in the promoter of c-MYC has been noted. On the other hand, association of NM23-H2 to the NHE leads to c-MYC activation. Therefore, NM23-H2 interaction with the G4 motif within the c-MYC NHE presents an interesting mechanistic possibility. Herein, using luciferase reporter assay and chromatin immunoprecipitation we show NM23-H2 mediated c-MYC activation involves NM23-H2-G4 motif binding within the c-MYC NHE. G4 motif complex formation with recombinant NM23-H2 was independently confirmed using fluorescence energy transfer, which also indicated that the G4 motif was resolved to an unfolded state within the protein-bound complex. Taken together, this supports transcriptional role of NM23-H2 via a G4 motif

Evidence of genome-wide G4 DNA-mediated gene expression in human cancer cells

Guanine-rich DNA of a particular sequence adopts four-stranded structural forms known as G-quadruplex or G4 DNA. Though in vitro formation of G4 DNA is known for several years, in vivo presence of G4 DNA was only recently noted in eukaryote telomeres. Recent bioinformatics analyses showing prevalence of G4 DNA within promoters of human and related species seems to implicate G4 DNA in a genome-wide cis-regulatory role. Herein we demonstrate that G4 DNA may present regulatory sites on a genome-wide scale by showing widespread effect on gene expression in response to the established intracellular G4 DNA-binding ligands. This is particularly relevant to genes that harbor conserved potential G4 DNA (PG4 DNA) forming sequence across human, mouse and rat promoters of orthologous genes. Genes with conserved PG4 DNA in promoters show co-regulated expression in 79 human and 61 mouse normal tissues (z-score > 3.5; P < 0.0001). Conservation of G4 DNA across related species also emphasizes the biological importance of G4 DNA and its role in transcriptional regulation of genes; shedding light on a relatively novel mechanism of regulation of gene expression in eukaryotes

A novel G-quadruplex motif modulates promoter activity of human thymidine kinase 1

G-quadruplex motifs constitute unusual DNA secondary structures formed by stacking of planar hydrogen-bonded G-tetrads. Recent genome-wide bioinformatics and experimental analyses have suggested the interesting possibility that G-quadruplex motifs could be cis-regulatory elements. Here, we identified a characteristic potential G-quadruplex-forming sequence element within the promoter of human thymidine kinase 1 (TK1). Our NMR, UV and CD spectroscopy and gel electrophoresis data suggested that this sequence forms a novel intramolecular G-quadruplex with two G-tetrads in K+ solution. The results presented here indicate the role of this G-quadruplex motif in transcription of TK1 in cell-based reporter assays. Specific nucleotide substitutions designed to destabilize the G-quadruplex motif resulted in increased promoter activity, supporting direct involvement of the G-quadruplex motif in transcription of TK1. These studies suggest that the G-quadruplex motif may be an important target for controlling critical biological processes, such as DNA synthesis, mediated by TK1

J Cell Sci

Cell migration is an essential and highly regulated process. During development, glia cells and neurons migrate over long distances - in most cases collectively - to reach their final destination and build the sophisticated architecture of the nervous system, the most complex tissue of the body. Collective migration is highly stereotyped and efficient, defects in the process leading to severe human diseases that include mental retardation. This dynamic process entails extensive cell communication and coordination, hence, the real challenge is to analyze it in the entire organism and at cellular resolution. We here investigate the impact of the N-cadherin adhesion molecule on collective glial migration, by using the Drosophila developing wing and cell-type specific manipulation of gene expression. We show that N-cadherin timely accumulates in glial cells and that its levels affect migration efficiency. N-cadherin works as a molecular brake in a dosage-dependent manner, by negatively controlling actin nucleation and cytoskeleton remodeling through alpha/beta catenins. This is the first in vivo evidence for N-cadherin negatively and cell autonomously controlling collective migration

Patient-matched analysis identifies deregulated networks in prostate cancer to guide personalized therapeutic intervention

International audienceProstate cancer (PrCa) is the second most common malignancy in men. More than 50% of advanced prostate cancers display the TMPRSS2-ERG fusion. Despite extensive cancer genome/transcriptome data, little is known about the impact of mutations and altered transcription on regulatory networks in the PrCa of individual patients. Using patient-matched normal and tumor samples, we established somatic variations and differential transcriptome profiles of primary ERG-positive prostate cancers. Integration of protein-protein interaction and generegulatory network databases defined highly diverse patient-specific network alterations. Different components of a given regulatory pathway were altered by novel and known mutations and/or aberrant gene expression, including deregulated ERG targets, and were validated by using a novel in silico methodology. Consequently, different sets of pathways were altered in each individual PrCa. In a given PrCa, several deregulated pathways share common factors, predicting synergistic effects on cancer progression. Our integrated analysis provides a paradigm to identify druggable key deregulated factors within regulatory networks to guide personalized therapies