Dysregulation of Translation Factors EIF2S1, EIF5A and EIF6 in Intestinal-Type Adenocarcinoma (ITAC)

Intestinal-type adenocarcinoma (ITAC) is a rare cancer of the nasal cavity and paranasal sinuses that occurs sporadically or secondary to exposure to occupational hazards, such as wood dust and leather. Eukaryotic translation initiation factors have been described as promising targets for novel cancer treatments in many cancers, but hardly anything is known about these factors in ITAC. Here we performed in silico analyses, evaluated the protein levels of EIF2S1, EIF5A and EIF6 in tumour samples and non-neoplastic tissue controls obtained from 145 patients, and correlated these results with clinical outcome data, including tumour site, stage, adjuvant radiotherapy and survival. In silico analyses revealed significant upregulation of the translation factors EIF6 (ITGB4BP), EIF5, EIF2S1 and EIF2S2 (p < 0.05) with a higher arithmetic mean expression in ITAC compared to non-neoplastic tissue (NNT). Immunohistochemical analyses using antibodies against EIF2S1 and EIF6 confirmed a significantly different expression at the protein level (p < 0.05). In conclusion, this work identifies the eukaryotic translation initiation factors EIF2S1 and EIF6 to be significantly upregulated in ITAC. As these factors have been described as promising therapeutic targets in other cancers, this work identifies candidate therapeutic targets in this rare but often deadly cancer.

The global gene expression outline of the bovine blastocyst: reflector of environmental conditions and predictor of developmental capacity

Background Morphological evaluation of embryos has been used to screen embryos for transfer. However, the repeatability and accuracy of this method remains low. Thus, evaluation of an embryo’s gene expression signature with respect to its developmental capacity could provide new opportunities for embryo selection. Since the gene expression outline of an embryo is considered as an aggregate of its intrinsic characteristics and culture conditions, we have compared transcriptome profiles of in vivo and in vitro derived blastocysts in relation to pregnancy outcome to unravel the discrete effects of developmental competence and environmental conditions on bovine embryo gene expression outlines. To understand whether the gene expression patterns could be associated with blastocyst developmental competency, the global transcriptome profile of in vivo (CVO) and in vitro (CVT) derived competent blastocysts that resulted in pregnancy was investigated relative to that of in vivo (NVO) and in vitro (NVT) derived blastocysts which did not establish initial pregnancy, respectively while to unravel the effects of culture condition on the transcriptome profile of embryos, the transcriptional activity of the CVO group was compared to the CVT group and the NVO group was compared to the NVT ones. Results A total of 700 differentially expressed genes (DEGs) were identified between CVO and NVO blastocysts. These gene transcripts represent constitutive regions, indel variants, 3′-UTR sequence variants and novel transcript regions. The majority (82%) of these DEGs, including gene clusters like ATP synthases, eukaryotic translation initiation factors, ribosomal proteins, mitochondrial ribosomal proteins, NADH dehydrogenase and cytochrome c oxidase subunits were enriched in the CVO group. These DEGs were involved in pathways associated with glycolysis/glycogenesis, citrate acid cycle, pyruvate metabolism and oxidative phosphorylation. Similarly, a total of 218 genes were differentially expressed between CVT and NVT groups. Of these, 89%, including TPT1, PDIA6, HSP90AA1 and CALM, were downregulated in the CVT group and those DEGs were overrepresented in pathways related to protein processing, endoplasmic reticulum, spliceasome, ubiquitone mediated proteolysis and steroid biosynthesis. On the other hand, although both the CVT and CVO blastocyst groups resulted in pregnancy, a total of 937 genes were differential expressed between the two groups. Compared to CVO embryos, the CVT ones exhibited downregulation of gene clusters including ribosomal proteins, mitochondrial ribosomal protein, eukaryotic translation initiation factors, ATP synthases, NADH dehydrogenase and cytochrome c oxidases. Nonetheless, downregulation of these genes could be associated with pre and postnatal abnormalities observed after transfer of in vitro embryos. Conclusion The present study provides a detailed inventory of differentially expressed gene signatures and pathways specifically reflective of the developmental environment and future developmental capacities of bovine embryos suggesting that transcriptome activity observed in blastocysts could be indicative of further pregnancy success but also adaptation to culture environment.

Dynamic competition between SARS-CoV-2 NSP1 and mRNA on the human ribosome inhibits translation initiation

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a beta-CoV that recently emerged as a human pathogen and is the causative agent of the COVID-19 pandemic. A molecular framework of how the virus manipulates host cellular machinery to facilitate infection remains unclear. Here, we focus on SARS-CoV-2 NSP1, which is proposed to be a virulence factor that inhibits protein synthesis by directly binding the human ribosome. We demonstrate biochemically that NSP1 inhibits translation of model human and SARS-CoV-2 messenger RNAs (mRNAs). NSP1 specifically binds to the small (40S) ribosomal subunit, which is required for translation inhibition. Using single-molecule fluorescence assays to monitor NSP1–40S subunit binding in real time, we determine that eukaryotic translation initiation factors (eIFs) allosterically modulate the interaction of NSP1 with ribosomal preinitiation complexes in the absence of mRNA. We further elucidate that NSP1 competes with RNA segments downstream of the start codon to bind the 40S subunit and that the protein is unable to associate rapidly with 80S ribosomes assembled on an mRNA. Collectively, our findings support a model where NSP1 proteins from viruses in at least two subgenera of beta-CoVs associate with the open head conformation of the 40S subunit to inhibit an early step of translation, by preventing accommodation of mRNA within the entry channel.

Eukaryotic translation initiation factors as promising targets in cancer therapy

The regulation of the translation of messenger RNA (mRNA) in eukaryotic cells is critical for gene expression, and occurs principally at the initiation phase which is mainly regulated by eukaryotic initiation factors (eIFs). eIFs are fundamental for the translation of mRNA and as such act as the primary targets of several signaling pathways to regulate gene expression. Mis-regulated mRNA expression is a common feature of tumorigenesis and the abnormal activity of eIF complexes triggered by upstream signaling pathways is detected in many tumors, leading to the selective translation of mRNA encoding proteins involved in tumorigenesis, metastasis, or resistance to anti-cancer drugs, and making eIFs a promising therapeutic target for various types of cancers. Here, we briefly outline our current understanding of the biology of eIFs, mainly focusing on the effects of several signaling pathways upon their functions and discuss their contributions to the initiation and progression of tumor growth. An overview of the progress in developing agents targeting the components of translation machinery for cancer treatment is also provided.

A small natural molecule CADPE kills residual colorectal cancer cells by inhibiting key transcription factors and translation initiation factors

Residual disease is the major cause for colorectal cancer (CRC) relapse. Herein, we explore whether and how a natural molecule CADPE killed heterogenic populations in a panel of CRC cell lines with KRAS/BRAF mutations that are natively resistant to EGFR- or VEGFR-targeted therapy, without sparing persistent cells, a reservoir of the disease relapse. Results showed that CADPE killed the tumor bulk and residual cells in the panel of CRC cell lines, rapidly inactivated c-Myc, STAT3, and NF-κB, and then decreased the protein levels of key signaling molecules for CRC, such as β-catenin, Notch1, and the nodes of mTOR pathways; eukaryotic translation initiation factors (eIF4F); anti-apoptotic proteins (Bcl-xl, Mcl-1, and survivin); and stemness-supporting molecules (CD133, Bim-1, and VEGF). In terms of mechanism of action, concurrent downregulation of Mcl-1, Bcl-xl, and survivin was necessary for CADPE to kill CRC bulk cells, while additional depletion of CD133 and VEGF proteins was required for killing the residual CRC cells. Moreover, the disabled c-Myc, STAT3, NF-κB, and eIF4F were associated with the broadly decreased levels of anti-apoptosis proteins and pro-stemness proteins. Consistently, CADPE suppressed CRC tumor growth associated with robust apoptosis and depleted levels of c-Myc, STAT3, NF-κB, eIF4F, anti-apoptotic proteins, and pro-stemness proteins. Our findings showed the promise of CADPE for treating CRC and suggested a rational polytherapy that disables c-Myc, STAT3, NF-κB, and eIF4F for killing CRC residual disease.

Dynamic competition between SARS-CoV-2 NSP1 and mRNA on the human ribosome inhibits translation initiation

ABSTRACTSARS-CoV-2 recently emerged as a human pathogen and is the causative agent of the COVID-19 pandemic. A molecular framework of how the virus manipulates host cellular machinery to facilitate infection remains unclear. Here, we focus on SARS-CoV-2 NSP1, which is proposed to be a virulence factor that inhibits protein synthesis by directly binding the human ribosome. Using extract-based and reconstitution experiments, we demonstrate that NSP1 inhibits translation initiation on model human and SARS-CoV-2 mRNAs. NSP1 also specifically binds to the small (40S) ribosomal subunit, which is required for translation inhibition. Using single-molecule fluorescence assays to monitor NSP1–40S subunit binding in real time, we demonstrate that eukaryotic translation initiation factors (eIFs) modulate the interaction: NSP1 rapidly and stably associates with most ribosomal pre-initiation complexes in the absence of mRNA, with particular enhancement and inhibition by eIF1 and eIF3j, respectively. Using model mRNAs and an inter-ribosomal-subunit FRET signal, we elucidate that NSP1 competes with RNA segments downstream of the start codon to bind the 40S subunit and that the protein is unable to associate rapidly with 80S ribosomes assembled on an mRNA. Collectively, our findings support a model where NSP1 associates with the open head conformation of the 40S subunit to inhibit an early step of translation, by preventing accommodation of mRNA within the entry channel.SIGNIFICANCE STATEMENTSARS-CoV-2 is the causative agent of the COVID-19 pandemic. A molecular framework for how SARS-CoV-2 manipulates host cellular machinery to facilitate infection is needed. Here, we integrate biochemical and single-molecule strategies to reveal molecular insight into how NSP1 from SARS-CoV-2 inhibits translation initiation. NSP1 directly binds to the small (40S) subunit of the human ribosome, which is modulated by human initiation factors. Further, NSP1 and mRNA compete with each other to bind the ribosome. Our findings suggest that the presence of NSP1 on the small ribosomal subunit prevents proper accommodation of the mRNA. How this competition disrupts the many steps of translation initiation is an important target for future studies.