scholarly journals TBIO-26. NON-CANONICAL OPEN READING FRAMES ENCODE FUNCTIONAL PROTEINS ESSENTIAL FOR CANCER CELL SURVIVAL

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii471-iii471
Author(s):  
John Prensner ◽  
Oana Enache ◽  
Victor Luria ◽  
Karsten Krug ◽  
Karl Clauser ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Protein Translation ◽  
Cancer Cell Lines ◽  
Open Reading Frames ◽  
Sequencing Analysis ◽  
Multiple Cancer ◽  
Cancer Types ◽  
Non Coding Rnas ◽  
Reading Frames

Abstract The brain is the foremost non-gonadal tissue for expression of non-coding RNAs of unclear function. Yet, whether such transcripts are truly non-coding or rather the source of non-canonical protein translation is unknown. Here, we used functional genomic screens to establish the cellular bioactivity of non-canonical proteins located in putative non-coding RNAs or untranslated regions of protein-coding genes. We experimentally interrogated 553 open reading frames (ORFs) identified by ribosome profiling for three major phenotypes: 257 (46%) demonstrated protein translation when ectopically expressed in HEK293T cells, 401 (73%) induced gene expression changes following ectopic expression across 4 cancer cell types, and 57 (10%) induced a viability defect when the endogenous ORF was knocked out using CRISPR/Cas9 in 8 human cancer cell lines. CRISPR tiling and start codon mutagenesis indicated that the biological impact of these non-canonical ORFs required their translation as opposed to RNA-mediated effects. We functionally characterized one of these ORFs, G029442—renamed GREP1 (Glycine-Rich Extracellular Protein-1)—as a cancer-implicated gene with high expression in multiple cancer types, such as gliomas. GREP1 knockout in >200 cancer cell lines reduced cell viability in multiple cancer types, including glioblastoma, in a cell-autonomous manner and produced cell cycle arrest via single-cell RNA sequencing. Analysis of the secretome of GREP1-expressing cells showed increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth inhibitory effect of GREP1 knock-out. Taken together, these experiments suggest that the non-canonical ORFeome is surprisingly rich in biologically active proteins and potential cancer therapeutic targets deserving of further study.

scholarly journals Non-canonical open reading frames encode functional proteins essential for cancer cell survival

2020 ◽  
Author(s):  
John R. Prensner ◽  
Oana M. Enache ◽  
Victor Luria ◽  
Karsten Krug ◽  
Karl R. Clauser ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Lines ◽  
Cancer Cell ◽  
Biological Effects ◽  
Protein Translation ◽  
Cancer Cell Lines ◽  
Open Reading Frames ◽  
Biologically Active ◽  
Knock Out ◽  
Reading Frames

A key question in genome research is whether biologically active proteins are restricted to the ∼20,000 canonical, well-annotated genes, or rather extend to the many non-canonical open reading frames (ORFs) predicted by genomic analyses. To address this, we experimentally interrogated 553 ORFs nominated in ribosome profiling datasets. Of these 553 ORFs, 57 (10%) induced a viability defect when the endogenous ORF was knocked out using CRISPR/Cas9 in 8 human cancer cell lines, 257 (46%) showed evidence of protein translation when ectopically expressed in HEK293T cells, and 401 (73%) induced gene expression changes measured by transcriptional profiling following ectopic expression across 4 cell types. CRISPR tiling and start codon mutagenesis indicated that the biological effects of these non-canonical ORFs required their translation as opposed to RNA-mediated effects. We selected one of these ORFs, G029442--renamed GREP1 (Glycine-Rich Extracellular Protein-1)--for further characterization. We found that GREP1 encodes a secreted protein highly expressed in breast cancer, and its knock-out in 263 cancer cell lines showed preferential essentiality in breast cancer derived lines. Analysis of the secretome of GREP1-expressing cells showed increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth inhibitory effect of GREP1 knock-out. Taken together, these experiments suggest that the non-canonical ORFeome is surprisingly rich in biologically active proteins and potential cancer therapeutic targets deserving of further study.

scholarly journals DNA-dependent protein synthesis exhibited by cancer shed particulates

2017 ◽  
Author(s):  
Vijay K. Ulaganathan ◽  
Axel Ullrich
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Ribosome Biogenesis ◽  
Protein Biosynthesis ◽  
Protein Translation ◽  
Cancer Cell Lines ◽  
Biologically Active ◽  
Multiple Cancer ◽  
Dependent Protein ◽  
Cancer Types

AbstractGenetic heterogeneity in tumours is the bonafide hallmark applicable to all cancer types (Burrell et al, 2013). Furthermore, deregulated ribosome biogenesis and elevated protein biosynthesis have been consistently associated with multiple cancer types (Ruggero, 2012; Ruggero & Pandolfi, 2003). We observed that under cultivation conditions almost all cancer cell types actively shed significant amount of particulates as compared to non-malignant cell lines requiring frequent changing of cultivation media. We therefore asked if cancer cell shed particulates might still retain biological activity associated with protein biosynthesis. Here, we communicate our observations of DNA-dependent protein biosynthetic activity exhibited by the cell-free particulates shed by the cancer cell lines. Using pulsed isotope labelling approach we confirmed the cell-free protein translation activity exhibited by particulates shed by various cancer cell lines. Interestingly, the bioactivity was largely dependent on temperature, pH and on 3’-DNA elements. Our results demonstrate that cancer shed particulates are biologically active and may potentially drive expression of tissue non-specific promoters in distant organs.

scholarly journals Integrating genetic dependencies and genomic alterations across pathways and cancer types

2020 ◽  
Author(s):  
Tae Yoon Park ◽  
Mark D.M. Leiserson ◽  
Gunnar W. Klau ◽  
Benjamin J. Raphael
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Synthetic Lethality ◽  
Cancer Cell Lines ◽  
Genetic Alterations ◽  
Cancer Type ◽  
Loss Of Function ◽  
Multiple Cancer ◽  
Genomic Alterations ◽  
Cancer Types

AbstractRecent genome-wide CRISPR-Cas9 loss-of-function screens have identified genetic dependencies across many cancer cell lines. Associations between these dependencies and genomic alterations in the same cell lines reveal phenomena such as oncogene addiction and synthetic lethality. However, comprehensive characterization of such associations is complicated by complex interactions between genes across genetically heterogeneous cancer types. We introduce SuperDendrix, an algorithm to identify differential dependencies across cell lines and to find associations between differential dependencies and combinations of genetic alterations and cell-type-specific markers. Application of SuperDendrix to CRISPR-Cas9 loss-of-function screens from 554 cancer cell lines reveals a landscape of associations between differential dependencies and genomic alterations across multiple cancer pathways in different combinations of cancer types. We find that these associations respect the position and type of interactions within pathways with increased dependencies on downstream activators of pathways, such as NFE2L2 and decreased dependencies on upstream activators of pathways, such as CDK6. SuperDendrix also reveals dozens of dependencies on lineage-specific transcription factors, identifies cancer-type-specific correlations between dependencies, and enables annotation of individual mutated residues.

scholarly journals Multi-Omics Analysis of Cancer Cell Lines with High/Low Ferroptosis Scores and Development of a Ferroptosis-Related Model for Multiple Cancer Types

2021 ◽  
Vol 9 ◽  
Author(s):  
Guangyao Shan ◽  
Huan Zhang ◽  
Guoshu Bi ◽  
Yunyi Bian ◽  
Jiaqi Liang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Cancer Cell ◽  
Gene Expression Data ◽  
Cancer Cell Lines ◽  
Expression Data ◽  
Rna Seq ◽  
Multiple Cancer ◽  
Related Model ◽  
Cancer Types

Background: Ferroptosis is a newly identified regulated cell death characterized by iron-dependent lipid peroxidation and subsequent membrane oxidative damage, which has been implicated in multiple types of cancers. The multi-omics differences between cancer cell lines with high/low ferroptosis scores remain to be elucidated.Methods and Materials: We used RNA-seq gene expression, gene mutation, miRNA expression, metabolites, copy number variation, and drug sensitivity data of cancer cell lines from DEPMAP to detect multi-omics differences associated with ferroptosis. Based on the gene expression data of cancer cell lines, we performed LASSO-Logistic regression analysis to build a ferroptosis-related model. Lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), esophageal cancer (ESCA), bladder cancer (BLCA), cervical cancer (CESC), and head and neck cancer (HNSC) patients from the TCGA database were used as validation cohorts to test the efficacy of this model.Results: After stratifying the cancer cell lines into high score (HS) and low score (LS) groups according to the median of ferroptosis scores generated by gene set variation analysis, we found that IC50 of 66 agents such as oxaliplatin (p < 0.001) were significantly different, among which 65 were higher in the HS group. 851 genes such as KEAP1 and NRAS were differentially muted between the two groups. Differentially expressed genes, miRNAs and metabolites were also detected—multiple items such as IL17F (logFC = 6.58, p < 0.001) differed between the two groups. Unlike the TCGA data generated by bulk RNA-seq, the gene expression data in DEPMAP are from pure cancer cells, so it could better reflect the traits of tumors in cancer patients. Thus, we built a 15-signature model (AUC = 0.878) based on the gene expression data of cancer cell lines. The validation cohorts demonstrated a higher mutational rate of NFE2L2 and higher expression levels of 12 ferroptosis-related genes in HS groups.Conclusion: This article systemically analyzed multi-omics differences between cancer cell lines with high/low ferroptosis scores and a ferroptosis-related model was developed for multiple cancer types. Our findings could improve our understanding of the role of ferroptosis in cancer and provide new insight into treatment for malignant tumors.

scholarly journals Species D Adenoviruses as Oncolytic Viral Vectors

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1399
Author(s):  
Brianna L. Bullard ◽  
Brigette N. Corder ◽  
Eric A. Weaver
Keyword(s):  
Blood Coagulation ◽  
Cell Lines ◽  
Cancer Cell ◽  
Neutralizing Antibodies ◽  
Coagulation Factor ◽  
Cancer Cell Lines ◽  
Factor X ◽  
Multiple Cancer ◽  
Iodide Uptake ◽  
Oncolytic Adenoviruses

Oncolytic adenoviruses (Ad) have shown promising results in the therapeutic treatment of cancer. Ad type 5 (Ad5) is the most extensively utilized Ad type. However, several limitations exist to using Ad5 as an oncolytic virus, including high levels of anti-Ad5 neutralizing antibodies in the population, binding of the Ad5 hexon to blood coagulation factor X leading to liver sequestration and toxicity, and reduced expression of the primary receptor CAR on many tumors. Here, we use in vitro methods to explore the oncolytic potential of four alternative Ad types (Ad26, 28, 45, and 48) belonging to the species D Ad subgroup and developed replication-competent species D Ads expressing the human sodium iodide symporter protein (hNIS) for combination radiovirotherapy. We evaluated the species D Ad vectors transduction, replication, cytotoxicity, and gene expression in six different cancer cell lines. Species D Ads showed the greatest transduction and cytotoxic killing in the SKBR3 breast cancer cells, followed by 293, A549, and HepG2 cells, however the cytotoxicity was less than the wild type Ad5 virus. In contrast, species D Ads showed limited transduction and cytotoxicity in the Hela and SKOV3 cancer cell lines. These species D Ad vectors also successfully expressed the hNIS gene during infection leading to increased iodide uptake in multiple cancer cell lines. These results, the low seroprevalence of anti-species D antibodies, and the lack of binding to blood coagulation FX, support further exploration of species D Ads as alternative oncolytic adenoviruses against multiple types of cancer.

scholarly journals An Integrated Bioinformatics Analysis Repurposes an Antihelminthic Drug Niclosamide for Treating HMGA2-Overexpressing Human Colorectal Cancer

Cancers ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 1482 ◽  
Author(s):  
Leung ◽  
Chou ◽  
Huang ◽  
Yang
Keyword(s):  
Colorectal Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Gene Signature ◽  
Cancer Cell Lines ◽  
Colorectal Cancer Cell ◽  
Sequencing Analysis ◽  
Colorectal Cancer Cells ◽  
Colorectal Cancer Cell Lines

Aberrant overexpression of high mobility group AT-hook 2 (HMGA2) is frequently found in cancers and HMGA2 has been considered an anticancer therapeutic target. In this study, a pan-cancer genomics survey based on Cancer Cell Line Encyclopedia (CCLE) and The Cancer Genome Atlas (TCGA) data indicated that HMGA2 was mainly overexpressed in gastrointestinal cancers including colorectal cancer. Intriguingly, HMGA2 overexpression had no prognostic impacts on cancer patients’ overall and disease-free survivals. In addition, HMGA2-overexpressing colorectal cancer cell lines did not display higher susceptibility to a previously identified HMGA2 inhibitor (netroposin). By microarray profiling of HMGA2-driven gene signature and subsequent Connectivity Map (CMap) database mining, we identified that S100 calcium-binding protein A4 (S100A4) may be a druggable vulnerability for HMGA2-overexpressing colorectal cancer. A repurposing S100A4 inhibitor, niclosamide, was found to reverse the HMGA2-driven gene signature both in colorectal cancer cell lines and patients’ tissues. In vitro and in vivo experiments validated that HMGA2-overexpressing colorectal cancer cells were more sensitive to niclosamide. However, inhibition of S100A4 by siRNAs and other inhibitors was not sufficient to exert effects like niclosamide. Further RNA sequencing analysis identified that niclosamide inhibited more cell-cycle-related gene expression in HMGA2-overexpressing colorectal cancer cells, which may explain its selective anticancer effect. Together, our study repurposes an anthelminthic drug niclosamide for treating HMGA2-overexpression colorectal cancer.

Anti-EpCAM XmAb antibodies with improved cytotoxicity

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 12506-12506 ◽  
Author(s):  
O. Vafa ◽  
S. Kharki ◽  
J. Vielmetter ◽  
A. Chamberlain ◽  
P. Hammond ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Clinical Efficacy ◽  
Cancer Cell Lines ◽  
In Vitro Cytotoxicity ◽  
Binding Assays ◽  
Multiple Cancer ◽  
Effector Cells ◽  
Dependent Cell

12506 Background: The epithelial cell adhesion molecule (EpCAM), also known as epithelial protein 2 (EGP-2) or 17–1A antigen, is a trans-membrane protein expressed on the surfaces of most carcinomas, including those of pancreatic, colorectal, prostate, breast, kidney, lung, and ovarian origins. Moderate affinity antibodies (Abs) such as 17–1A (Kd ∼ 10−7 nM) have been safe in humans albeit with limited clinical efficacy. Attempts to improve clinical efficacy by enhancing antigen affinity (Kd ∼ 10−9 nM) have led to serious clinical toxicity, including pancreatitis. These observations raise the question of whether a moderate affinity Ab with enhanced effector function will be both safe and clinically efficacious. Methods: We applied our proprietary XmAb™ technologies to humanize the 17–1A variable domain and engineer a human IgG1 Fc domain to increase affinity for the activating receptor FcγRIIIa. Ab binding to Ep-CAM or to Fc receptors was tested with Biacore and/or AlphaScreen binding assays. In vitro cytotoxic activity against representative cancer cell lines was measured with Antibody Dependent Cell-mediated Cytotoxicity (ADCC) assays, using human PBMC as effector cells. Results: Humanized anti-EpCAM Abs have affinity for EpCAM similar to the parent 17–1A. Affinity for the activating FcγRIIIa was increased 100-fold relative to a control Ab with an IgG1 Fc domain. As expected, these Abs exhibit dramatically enhanced ADCC against multiple cancer cell lines relative to 17–1A and IgG1 control Abs. Despite their moderate affinity for EpCAM, these novel Abs have in vitro cytotoxicity comparable to the high affinity Ab ING-1. CDC activities of these Abs were similar to chimeric 17–1A. Conclusions: We have demonstrated that antibodies with moderate affinity for EpCAM and increased FcγRIIIa affinity exhibit superior cancer cell killing via an ADCC mechanism. The humanized nature and the increased cytotoxicity of anti-EpCAM XmAb™ antibodies make them promising candidates for clinical development of a novel pan-carcinoma Ab that is superior to 17–1A. [Table: see text]

scholarly journals Extracting Biological Insights from the Project Achilles Genome-Scale CRISPR Screens in Cancer Cell Lines

2019 ◽  
Author(s):  
Joshua M. Dempster ◽  
Jordan Rossen ◽  
Mariya Kazachkova ◽  
Joshua Pan ◽  
Guillaume Kugener ◽  
...  
Keyword(s):  
Data Processing ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Loss Of Function ◽  
The Public ◽  
Quality Control Metrics ◽  
Cancer Types ◽  
Genome Scale

AbstractOne of the main goals of the Cancer Dependency Map project is to systematically identify cancer vulnerabilities across cancer types to accelerate therapeutic discovery. Project Achilles serves this goal through the in vitro study of genetic dependencies in cancer cell lines using CRISPR/Cas9 (and, previously, RNAi) loss-of-function screens. The project is committed to the public release of its experimental results quarterly on the DepMap Portal (https://depmap.org), on a pre-publication basis. As the experiment has evolved, data processing procedures have changed. Here we present the current and projected Achilles processing pipeline, including recent improvements and the analyses that led us to adopt them, spanning data releases from early 2018 to the first quarter of 2020. Notable changes include quality control metrics, calculation of probabilities of dependency, and correction for screen quality and other biases. Developing and improving methods for extracting biologically-meaningful scores from Achilles experiments is an ongoing process, and we will continue to evaluate and revise data processing procedures to produce the best results.

scholarly journals Design, Synthesis and Anticancer Profile of New 4-(1H-benzo[d]imidazol-1-yl)pyrimidin-2-amine-Linked Sulfonamide Derivatives with V600EBRAF Inhibitory Effect

2021 ◽  
Vol 22 (19) ◽  
pp. 10491
Author(s):  
Mohammed S. Abdel-Maksoud ◽  
Ahmed A. B. Mohamed ◽  
Rasha M. Hassan ◽  
Mohamed A. Abdelgawad ◽  
Garri Chilingaryan ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cell Cycle Progression ◽  
Pyrimidine Ring ◽  
Cancer Cell Lines ◽  
Inhibitory Effect ◽  
Docking Studies ◽  
Binding Modes ◽  
Multiple Cancer

A new series of 4-(1H-benzo[d]imidazol-1-yl)pyrimidin-2-amine linked sulfonamide derivatives 12a–n was designed and synthesized according to the structure of well-established V600EBRAF inhibitors. The terminal sulfonamide moiety was linked to the pyrimidine ring via either ethylamine or propylamine bridge. The designed series was tested at fixed concentration (1 µM) against V600EBRAF, finding that 12e, 12i and 12l exhibited the strongest inhibitory activity among all target compounds and 12l had the lowest IC50 of 0.49 µM. They were further screened on NCI 60 cancer cell lines to reveal that 12e showed the most significant growth inhibition against multiple cancer cell lines. Therefore, cell cycle analysis of 12e was conducted to investigate the effect on cell cycle progression. Finally, virtual docking studies was performed to gain insights for the plausible binding modes of vemurafenib, 12i, 12e and 12l.

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