Trichostatin A preferentially reverses the upregulation of gene-expression levels induced by gain of chromosome 7 in colorectal cancer cell lines.

Epithelial cancers are defined by a tumor-specific distribution of chromosomal aneuploidies that are maintained when cells metastasize and are conserved in cell lines derived from primary tumors. Correlations between genomic copy number and gene expression have been observed for different tumors including, colorectal (CRC), breast, and pancreatic cancer. These ploidy-driven transcriptional deregulations are characterized by low-level expression changes of most genes on the affected chromosomes. The emergence of these aberrations at an early stage of tumorigenesis and the strong selection for the maintenance of these aneuploidies suggest that aneuploidy-dependent transcriptional deregulations might contribute to cellular transformation and maintenance of the malignant phenotype. The histone deacetylase inhibitor (HDACi) Trichostatin A (TSA) has anticancer effects and is well known to lead to large-scale gene-expression changes. Here we assessed if TSA could disrupt the aneuploidy-driven gene expression in the aneuploid colon cancer cell line SW480 and the artificially generated aneuploid cell line DLD-1 + 7. We found that TSA increases transcriptional activity throughout the genome, yet inhibits aneuploidy-induced gene-expression changes on chromosome 7. Among the TSA affected genes on chromosome 7, we identified potential CRC oncogenes. These experiments represent the first attempt to explain how histone acetylation affects aneuploidy-driven gene-expression changes.

Aneuploidy, oncogene amplification and epithelial to mesenchymal transition define spontaneous transformation of murine epithelial cells.

Human epithelial cancers are defined by a recurrent distribution of specific chromosomal aneuploidies, a trait less typical for murine cancer models induced by an oncogenic stimulus. After prolonged culture, mouse epithelial cells spontaneously immortalize, transform and become tumorigenic. We assessed genome and transcriptome alterations in cultures derived from bladder and kidney utilizing spectral karyotyping, array CGH, FISH and gene expression profiling. The results show widespread aneuploidy, yet a recurrent and tissue-specific distribution of genomic imbalances, just as in human cancers. Losses of chromosome 4 and gains of chromosome 15 are common and occur early during the transformation process. Global gene expression profiling revealed early and significant transcriptional deregulation. Chromosomal aneuploidy resulted in expression changes of resident genes and consequently in a massive deregulation of the cellular transcriptome. Pathway interrogation of expression changes during the sequential steps of transformation revealed enrichment of genes associated with DNA repair, centrosome regulation, stem cell characteristics and aneuploidy. Genes that modulate the epithelial to mesenchymal transition and genes that define the chromosomal instability phenotype played a dominant role and were changed in a directionality consistent with loss of cell adhesion, invasiveness and proliferation. Comparison with gene expression changes during human bladder and kidney tumorigenesis revealed remarkable overlap with changes observed in the spontaneously transformed murine cultures. Therefore, our novel mouse models faithfully recapitulate the sequence of genomic and transcriptomic events that define human tumorigenesis, hence validating them for both basic and preclinical research.

Nck-1 interacts with PKR and modulates its activation by dsRNA.

Activation of the double-stranded RNA (dsRNA)-activated protein kinase PKR results in inhibition of general translation through phosphorylation of the eukaryotic initiation factor 2 alpha-subunit on serine 51 (eIF2alphaSer51). Previously, we have reported that the adaptor protein Nck-1 modulates eIF2alphaSer51 phosphorylation by a subset of eIF2alpha kinases, including PKR. Herein, we demonstrate that Nck-1 prevents efficient activation of PKR by dsRNA, revealing that Nck-1 acts at the level of PKR. In agreement, Nck-1 impairs p38MAPK activation and attenuates cell death induced by dsRNA, in addition to diminish eIF2alphaSer51 phosphorylation. Our data show that the inhibitory effect of Nck-1 on PKR is reversible, as it could be overcome by increasing levels of dsRNA. Interestingly, we found that Nck-1 interacts with the inactive form of PKR, independently of its Src homology domains. Furthermore, we uncovered that Nck-1 is substrate of PKR in vitro. All together, our data provide the first evidence identifying Nck-1 as a novel endogenous regulator of PKR and support the notion that Nck-1-PKR interaction could be a way to limit PKR activation.

Nck-1 selectively modulates eIF2alphaSer51 phosphorylation by a subset of eIF2alpha-kinases.

Phosphorylation of the alpha-subunit of the eukaryotic initiation factor 2 (eIF2) on Ser51 is an early event associated with the down-regulation of protein synthesis at the level of translation and initiation of a transcriptional program. This constitutes a potent mechanism to overcome various stress conditions. In mammals, four eIF2alpha-kinases [PKR-like endoplasmic reticulum kinase (PERK), dsRNA-activated protein kinase (PKR), heme regulated inhibitor (HRI) and general control nonderepressible-2 (GCN2)], activated following specific stresses, have been shown to be involved in this process. In this article, we report that the ubiquitously expressed adaptor protein Nck, composed only of Src homology domains and classically implicated in cell signaling by activated plasma membrane receptor tyrosine kinases, modulates eIF2alpha-kinase-mediated eIF2alphaSer51 phosphorylation in a specific manner. Our results show that Nck not only prevents eIF2alpha phosphorylation upon PERK activation, as reported previously, but also reduces eIF2alpha phosphorylation in conditions leading to PKR and HRI activation. By contrast, the overexpression of Nck in mammalian cells fails to attenuate eIF2alphaSer51 phosphorylation in response to amino acid starvation, a stress well known to activate GCN2. This observation is further confirmed by showing that Nck fails to alter eIF2alphaSer51 phosphorylation in Saccharomyces cerevisiae, for which the sole eIF2alpha-kinase is Gcn2p. Our results suggest the existence of a novel mechanism that specifically modulates the phosphorylation of eIF2alpha on Ser51 under various stress conditions.

Nck-1 antagonizes the endoplasmic reticulum stress-induced inhibition of translation.

Eukaryotic cells have developed specific mechanisms to overcome environmental stress. Here we show that the Src homology 2/3 (SH2/SH3) domain-containing protein Nck-1 prevents the unfolded protein response normally induced by pharmacological endoplasmic reticulum (ER) stress agents. Overexpression of Nck-1 enhances protein translation, whereas it abrogates eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation and inhibition of translation in response to tunicamycin or thapsigargin treatment. Nck-1 overexpression also attenuates induction of the ER chaperone, the immunoglobulin heavy chain-binding protein (BiP), and impairs cell survival in response to thapsigargin. We provided evidence that in these conditions, the effects of Nck on the unfolded protein response (UPR) involve its second SH3 domain and a calyculin A-sensitive phosphatase activity. In addition, we demonstrated that protein translation is reduced in mouse embryonic fibroblasts lacking both Nck isoforms and is enhanced in similar cells expressing high levels of Nck-1. In these various mouse embryonic fibroblasts, we also provided evidence that Nck modulates the activation of the ER resident eIF2alpha kinase PERK and consequently the phosphorylation of eIF2alpha on Ser-51 in response to stress. Our study establishes that Nck is required for optimal protein translation and demonstrates that, in addition to its adaptor function in mediating signaling from the plasma membrane, Nck also mediates signaling from the ER membrane compartment.