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 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 Evaluating Expression of the STAG1 Gene as a Potential Breast Cancer Biomarker

2021 ◽  
Vol 45 (2) ◽  
pp. 7-13
Author(s):  
Inam J Lafta ◽  
Bassam K Kudhair ◽  
Oluyinka A Iyiola ◽  
Emad A Ahmed ◽  
Tachung Chou
Keyword(s):  
Breast Cancer ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cell Biology ◽  
Cancer Cell Lines ◽  
Eukaryotic Cell ◽  
Normal Breast ◽  
Cancer Biomarker ◽  
Open Reading Frames ◽  
Breast Cancer Cell Lines

STAG proteins, which are part of the cohesin complex and encoded by the STAG genes, are known as Irr1/Scc3 in yeast and as SA/STAG/stromalin in mammals. There are more variants as there are alternate splice sites, maybe three open reading frames (ORFs) code for three main proteins, including: SA1 (STAG1), SA2 (STAG2) and SA3 (STAG3). The cohesin protein complex has various essential roles in eukaryotic cell biology. This study compared the expression of the STAG1 gene in four different breast cancer cell lines, including: MCF-7, T-47D, MDA-MB-468, and MDA-MB-231 and normal breast tissue. RNA was extracted from these cell lines and mRNA was converted to cDNA, and then expression of the STAG1 gene was quantified by three sets of specific primer pairs using Real Time-quantitative PCR (RT-qPCR). The findings show significantly different over-expression of STAG1 in these cancer cell lines in comparison with the normal tissue, and the cell lines were different in their expression levels. In conclusion, the STAG1 gene can be postulated as a candidate breast cancer biomarker that needs to be further evaluated in breast tumor biopsies.

scholarly journals Jujuboside B Inhibits the Proliferation of Breast Cancer Cell Lines by Inducing Apoptosis and Autophagy

2021 ◽  
Vol 12 ◽  
Author(s):  
Lin Guo ◽  
Yupei Liang ◽  
Shiwen Wang ◽  
Lihui Li ◽  
Lili Cai ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Lines ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Cancer Cell Lines ◽  
Biologically Active ◽  
Breast Cancer Cell Lines ◽  
Induced Apoptosis ◽  
Mcf 7

Jujuboside B (JB) is one of the main biologically active ingredients extracted from Zizyphi Spinosi Semen (ZSS), a widely used traditional Chinese medicine for treating insomnia and anxiety. Breast cancer is the most common cancer and the second leading cause of cancer-related death in women worldwide. The purpose of this study was to examine whether JB could prevent breast cancer and its underlying mechanism. First, we reported that JB induced apoptosis and autophagy in MDA-MB-231 and MCF-7 human breast cancer cell lines. Further mechanistic studies have revealed that JB-induced apoptosis was mediated by NOXA in both two cell lines. Moreover, the AMPK signaling pathway plays an important role in JB-induced autophagy in MCF-7. To confirm the anti-breast cancer effect of JB, the interaction of JB-induced apoptosis and autophagy was investigated by both pharmacological and genetic approaches. Results indicated that autophagy played a pro-survival role in attenuating apoptosis. Further in vivo study showed that JB significantly suppressed the growth of MDA-MB-231 and MCF-7 xenografts. In conclusion, our findings indicate that JB exerts its anti-breast cancer effect in association with the induction of apoptosis and autophagy.

scholarly journals BIOL-03. PROTEIN TRANSLATION FROM NON-CODING GENOMIC LOCI PRODUCE BIOLOGICALLY-ACTIVE PROTEINS IMPLICATED IN CANCER CELL SURVIVAL IN PEDIATRIC BRAIN TUMORS

2021 ◽  
Vol 23 (Supplement_1) ◽  
pp. i3-i3
Author(s):  
John Prensner ◽  
Todd Golub
Keyword(s):  
Brain Tumors ◽  
Cell Survival ◽  
Cell Lines ◽  
Cancer Cell ◽  
Biological Effects ◽  
Protein Translation ◽  
Pediatric Brain Tumors ◽  
Biologically Active ◽  
Cancer Cell Survival ◽  
Pediatric Brain

Abstract Protein translation is both a fundamental cellular process essential for life as well as an oncogenic mechanism employed by tumors to enact cancer cell biology. While protein translation is most readily manifest in the ~20,000 known human protein coding genes, there are, in fact, several thousand additional regions of the cancer genome that are translated and contribute the complexity of the molecular milieu of cancer. Here, we systematically addressed the question of whether such uncharacterized genomic regions encode truly biologically active proteins and applied these findings to pediatric brain tumors. We experimentally interrogated 553 candidates selected from non-canonical open reading frame (ORF) datasets. Of these, 57 induced viability defects when knocked out in a broad array of human cancer cell lines. Upon ectopic expression, 257 showed evidence of protein expression and 401 induced gene expression changes. CRISPR tiling and start codon mutagenesis indicated that their biological effects required translation as opposed to RNA-mediated effects. We characterized several of these in the context of pediatric brain tumors, where dense CRISPR tiling screens revealed unique functional relevance of dozens of non-canonical ORFs in pediatric brain cancer cell survival. We found that one of these ORFs, ASNSD1 uORF, encodes a well-folded protein whose translation is a selective genetic dependency distinct from the adjacent ASNSD1 annotated protein. In vitro molecular biology assays confirmed the MYC-amplified medulloblastoma cell lines had a heightened dependency on this protein, and that MYC binds to the promoter of this gene, with MYC expression correlating with ASNSD1 in patient tumors. Co-immunoprecipitation assays defined ASNSD1 uORF as a novel member of the prefoldin complex of cytoplasmic protein stability regulators. Overall, our experiments suggest that the abundant protein translation found in the “non-coding” genome may produce biologically active non-canonical ORFs that are potential therapeutic targets.

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 Cytotoxicity and Apoptosis Inducing Effects of Some Lathyrane and Tigliane Diterpenes against Breast Cancer Cell Lines

2021 ◽  
Author(s):  
Melika Maleki ◽  
Zeinab Yazdiniapour ◽  
Mustafa Ghanadian ◽  
Behzad Zolfaghari ◽  
Faezeh Rabbani ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Lines ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Biological Effects ◽  
Cancer Cell Lines ◽  
Breast Cancer Cell Lines ◽  
Cytotoxic Effects ◽  
Apoptotic Effects ◽  
Mcf 7

Natural compounds and especially herbal medicine are of great interest due to their various biological effects and their potential to act as a drug for the treatment of various neoplasms especially breast cancer that we are facing with its increasing prevalence around the world. The aim of this study was to evaluate the cytotoxic and cell death mechanism of some diterpenoids (Lathyrane or Tigliane) extracted from the Euphorbia sogdiana Popov against two breast cancer cell lines, MCF-7 and 4T1. Determination the cytotoxic effects of four various diterpenoids was performed using MTT assay against MCF-7, 4T1, and HUVEC cell lines. The IC50 of each compound against cell lines was determined by drawing the dose-response graph using graph Pad prism software. Finally, the apoptotic effects of compound with the most cytotoxic effects was determined by flow cytometry assay for 24 hrs of incubation in IC50 concentration. Statistical analysis confirmed compound (3) with the most cytotoxicity against both cancer cell lines. The IC50 of compound (3) was determined as 10.1 ± 5, 28 ± 5, and 50 ± 3 µg/ml, for MCF-7, 4T1, and HUVEC cells, respectively. Furthermore, the cells treated with 5 and 10 μg/ml of compound (3) for 24 hrs, showed 49 and 57% of apoptosis. These surveyed compounds have the potential to be considered as useful anti-breast cancer agents due to the great cytotoxicity and apoptotic effects against related cancer cell lines and safety profile according to their rational selectivity index.

Cytotoxic activity and anti-cancer potential of Ontario grown onion extracts against breast cancer cell lines

2018 ◽  
Vol 8 (3) ◽  
pp. 159 ◽  
Author(s):  
Meghan Fragis ◽  
Abdulmonem I. Murayyan ◽  
Suresh Neethirajan
Keyword(s):  
Breast Cancer ◽  
Cytotoxic Activity ◽  
Cell Lines ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Cancer Cell Lines ◽  
Breast Cancer Cell Lines ◽  
Cytotoxic Activities ◽  
Cancer Line ◽  
Mcf 7

Background: Breast cancer is the most commonly diagnosed cancer and the second leading cause of cancer deaths among Canadian women. Cancer management through changes in lifestyle, such as increased intake of foods rich in dietary flavonoids, have been shown to decrease the risk associated with breast, liver, colorectal, and upper-digestive cancers in epidemiologic studies. Onions are high in flavonoid content and one of the most common vegetables. Additionally, onions are used in most Canadian cuisines.Methods: We investigated the effect of five prominent Ontario grown onion (Stanley, Ruby Ring, LaSalle, Fortress, and Safrane) extracts on two subtypes of breast cancer cell lines: a triple negative breast cancer line MDA-MB-231 and an ER+ breast cancer line MCF-7.Results: These onion extracts elicited strong anti-proliferative, anti-migratory, and cytotoxic activities on both the cancer cell lines. Flavonoids present in these onion extracts induced apoptosis, cell cycle arrest in the G2/M phase, and a reduction in mitochondrial membrane potential at dose-dependent concentrations. Onion extracts were more effective against MDA-MB-231 compared to the MCF-7 cell line. Conclusion: In this study, we investigated the extracts synthesized from Ontario-grown onion varieties in inducing anti-migratory, cytostatic, and cytotoxic activities in two sub-types of human breast cancer cell lines. Anti-tumor activity of these extracts depends upon the varietal and can be formulated into nutraceuticals and functional foods for the wellbeing of cancer patients. Overall, the results suggest that onion extracts are a good source of flavonoids with anti-cancerous properties.Keywords: onion extracts; flavonoids; anti-proliferative; breast cancer; cytotoxic activity

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