Ribosomal Protein Synthesis Research Articles

Distinct effects of CDK8 module subunits on cellular growth and proliferation in Drosophila.

The Mediator complex plays a pivotal role in facilitating RNA polymerase II-dependent transcription in eukaryotes. Within this complex, the CDK8 kinase module (CKM), comprising CDK8, Cyclin C (CycC), Med12, and Med13, serves as a dissociable subcomplex that modulates the activity of the small Mediator complex. Genetic studies in Drosophila have revealed distinct phenotypes associated with mutations in CKM subunits, but the underlying mechanisms have remained unclear. Using Drosophila as a model, we generated transgenic strains to individually or simultaneously deplete the four CKM subunits in all possible combinations, uncovering unique phenotypes in the eyes and wings. Depletion of CDK8-CycC enhanced E2F1 target gene expression and promoted cell-cycle progression, whereas Med12-Med13 depletion had no significant impact on these processes. Instead, depleting Med12-Med13 altered the expression of ribosomal protein genes and fibrillarin, reduced nascent protein synthesis, indicating a severe reduction in ribosome biogenesis and cellular growth compared to the loss of CDK8-CycC. These findings reveal distinct in vivo roles for CKM subunits, with Med12-Med13 disruption having a more pronounced effect on ribosome biogenesis and protein synthesis than CDK8-CycC loss.

The Interplay Between the MYC Oncogene and Ribosomal Proteins in Osteosarcoma Onset and Progression: Potential Mechanisms and Indication of Candidate Therapeutic Targets

High-grade osteosarcoma (OS) is the most common primary bone tumor mainly affecting children and young adults. First-line treatment consists of neo-adjuvant chemotherapy with doxorubicin, cisplatin, and methotrexate and surgery. The mean long-term survival rate for localized disease at diagnosis is 65–70%, dropping down to 20% when metastases are present at diagnosis. Therefore, curing OS is a clinical challenge, particularly for patients that do not respond to standard treatments. MYC has frequently been reported to be involved in the pathogenesis of OS and its high expression may be associated with drug resistance and patients’ worse prognosis. Moreover, MYC is a master regulator of ribosomal proteins (RPs) synthesis and ribosome biogenesis (RiBi), which is often up-regulated in human tumors. In recent years, RPs have been recognized not only for their traditional role in ribosome assembly but also for their extra-ribosomal functions, many of which are linked to the onset and progression of cancer. In this review we focus on the role and possible interplay of MYC and RPs expression in association with drug resistance and worse prognosis in OS and discuss therapeutic options that target de-regulated MYC, RiBi, or RPs, which are already clinically available or under evaluation in clinical trials.

Microplastic-Enhanced Cadmium Toxicity: A Growing Threat to the Sea Grape, Caulerpa lentillifera.

The escalating impact of human activities has led to the accumulation of microplastics (MPs) and heavy metals in marine environments, posing serious threats to marine ecosystems. As essential components of oceanic ecosystems, large seaweeds such as Caulerpa lentillifera play a crucial role in maintaining ecological balance. This study investigated the effects of MPs and cadmium (Cd) on the growth, physiology, biochemistry, and Cd accumulation in C. lentillifera while elucidating the underlying molecular regulatory mechanisms. The results demonstrated that exposure to MPs alone significantly promoted the growth. In contrast, exposure to Cd either alone or in combination with MPs significantly suppressed growth by reducing stem and stolon length, bud count, weight gain, and specific growth rates. Combined exposure to MPs and Cd exhibited the most pronounced inhibitory effect on growth. MPs had negligible impact while Cd exposure either alone or combined with MPs impaired antioxidant defenses and exacerbated oxidative damage; with combined exposure being the most detrimental. Analysis of Cd content revealed that MPs significantly increased Cd accumulation in algae intensifying its toxic effects. Gene expression analysis revealed that Cd exposure down-regulated key genes involved in photosynthesis, impairing both photosynthetic efficiency and energy conversion. The combined exposure of MPs and Cd further exacerbated these effects. In contrast, MPs alone activated the ribosome pathway, supporting ribosomal stability and protein synthesis. Additionally, both Cd exposure alone or in combination with MPs significantly reduced chlorophyll B and soluble sugar content, negatively impacting photosynthesis and nutrient accumulation. In summary, low concentrations of MPs promoted C. lentillifera growth, but the presence of Cd hindered it by disrupting photosynthesis and antioxidant mechanisms. Furthermore, the coexistence of MPs intensified the toxic effects of Cd. These findings enhance our understanding of how both MPs and Cd impact large seaweed ecosystems and provide crucial insights for assessing their ecological risks.

199 Awardee Talk: Insights into translational regulation through tRNA sequencing, RNA sequencing, and ribosome profiling

Abstract Translation acts as an additional layer of regulation that has an important role in gene expression and function. Due to a phenomenon called codon usage bias, highly expressed genes are thought to be codon-biased to support efficient translation. As more than one codon can code for the same amino acid (synonymous codons), organisms may exhibit preferences for specific codons that facilitate increased expression of important genes due to variations in the availability of corresponding tRNAs. Advances in sequencing technologies have recently permitted the study of the translatome, which refers to the entire population of mRNA associated with ribosomes for protein synthesis and can be investigated through ribosome profiling. This cutting-edge technique allows the identification of actively translated regions, but can also reveal translational pausing events that stem from the presence of SNPs. Our previous research has demonstrated variation in tRNA expression across tissues and different states of health, leading us to consider the connection between tRNA abundance and translational stalling due to SNPs. By coupling ribosome profiling with tRNA sequencing and RNA sequencing, we have investigated all elements of translational machinery to predict translational efficiency, estimate proteome composition, and evaluate tRNA abundance as a source of genetic variation. In this work, we utilized ribosome profiling, tRNAseq, and RNAseq and performed an integrative analysis in bovine tissues (kidney, liver and muscle) as well as murine myoblast cell lines (C2C12). By applying these methods to different bovine tissues, we were able to explore the interplay between tRNA availability and translational stalling events. Moreover, we have identified translationally regulated genes underlying tissue-specific biological processes and found that many upregulated and downregulated genes coincided with high and low translational efficiency respectively. We have also successfully defined stalling sites that depict the regulatory information encoded within the coding sequence of transcripts, which could control translation rate and facilitate proper protein folding. Through the implementation of these next generation sequencing (NGS) methods to cell culture, we were able to evaluate the translatome at various time points (0-min, 30-min, 60-min, and 4-h) post-induction of differentiation. Where most studies have focused on molecular changes between differentiating myoblasts and multinucleated myotubes that are present 7 d after differentiation, we focus on the earliest stages of muscle differentiation that initiate muscle development and therefore kickstart the process of meat production. This work offers an atlas of distinctive stalling sites across bovine tissues and various stages of muscle differentiation, which provides an opportunity to predict codon optimality and understand tissue-specific or time-specific mechanisms of regulating protein synthesis.

USP4 regulates ribosome biogenesis and protein synthesis for hematopoietic stem cell regeneration and leukemia progression.

Enhanced ribosome biogenesis and protein synthesis are required for cell proliferation. During hematopoietic regeneration, hematopoietic stem cells (HSCs) proliferate rapidly to replenish the hematopoietic system. How HSCs respond and regulate ribosome biogenesis and protein synthesis during regeneration remains unclear. Here, we analyzed the expression of a series of ubiquitin-specific-proteases (USPs) during HSC regeneration. We found USP4 expression is significantly increased in proliferating HSCs. Further functional and mechanistic investigations revealed a crucial regulatory function of USP4 in HSC regeneration and leukemia progression by modulating ribosome biogenesis and protein synthesis. USP4 deubiquitinates and stabilizes PES1 to facilitate ribosome biogenesis and protein synthesis in proliferative HSCs and leukemic cells. Usp4 deletion significantly decreases protein synthesis, proliferation and reconstitution capacity of HSCs. Usp4 inhibition suppresses ribosome biogenesis and proliferation of leukemic cells, and prolongs the survival of AML (Acute myeloid leukemia) mice. These findings provide a new insight into the response mechanism of ribosome biogenesis and protein synthesis in HSCs, and their contribution to leukemia progression.

Synthesis at the Interface of Chemistry and Biology.

ConspectusChemical synthesis as a tool to control the structure and properties of matter is at the heart of chemistry─from the synthesis of fine chemicals and polymers to drugs and solid-state materials. But as the field evolves to tackle larger and larger molecules and molecular complexes, the traditional tools of synthetic chemistry become limiting. In contrast, Mother Nature has developed very different strategies to create the macromolecules and molecular systems that make up the living cell. Our focus has been to ask whether we can use the synthetic strategies and machinery of Mother Nature, together with modern chemical tools, to create new macromolecules, and even whole organisms with properties not existing in nature. One such example involves reprogramming the complex, multicomponent machinery of ribosomal protein synthesis to add new building blocks to the genetic code, overcoming a billion-year constraint on the chemical nature of proteins. This methodology exploits the concept of bioorthogonality to add unique codons, tRNAs and aminoacyl-tRNA synthetases to cells to encode amino acids with physical, chemical and biological properties not found in nature. As a result, we can make precise changes to the structures of proteins, much like those made by chemists to small molecules and beyond those possible by biological approaches alone. This technology has made it possible to probe protein structure and function in vitro and in vivo in ways heretofore not possible, and to make therapeutic proteins with enhanced pharmacology. A second example involves exploiting the molecular diversity of the humoral immune system together with synthetic transition state analogues to make catalytic antibodies, and then expanding this diversity-based strategy (new to chemists at the time) to drug discovery and materials science. This work ushered in a new nature-inspired synthetic strategy in which large libraries of natural or synthetic molecules are designed and then rationally selected or screened for new function, increasing the efficiency by which we can explore chemical space for new physical, chemical and biological properties. A final example is the use of large chemical libraries, robotics and high throughput phenotypic cellular screens to identify small synthetic molecules that can be used to probe and manipulate the complex biology of the cell, exemplified by druglike molecules that control cell fate. This approach provides new insights into complex biology that complements genomic approaches and can lead to new drugs that act by novel mechanisms of action, for example to selectively regenerate tissues. These and other advances have been made possible by using our knowledge of molecular structure and reactivity hand in hand with our understanding of and ability to manipulate the complex machinery of living cells, opening a new frontier in synthesis. This Account overviews the work in my lab and with our collaborators, from our early days to the present, that revolves around this central theme.

Enhanced binding of guanylated poly(A) RNA by the LaM domain of LARP1

ABSTRACT La-related proteins (LARPs) are a family of RNA-binding proteins that share a conserved La motif (LaM) domain. LARP1 plays a role in regulating ribosomal protein synthesis and stabilizing mRNAs and has a unique structure without an RNA binding RRM domain adjoining the LaM domain. In this study, we investigated the physical basis for LARP1 specificity for poly(A) sequences and observed an unexpected bias for sequences with single guanines. Multiple guanine substitutions did not increase the affinity, demonstrating preferential recognition of singly guanylated sequences. We also observed that the cyclic di-nucleotides in the cCAS/STING pathway, cyclic-di-GMP and 3‘,3’-cGAMP, bound with sub-micromolar affinity. Isothermal titration measurements were complemented by high-resolution crystal structures of the LARP1 LaM with six different RNA ligands, including two stereoisomers of a phosphorothioate linkage. The selectivity for singly substituted poly(A) sequences suggests LARP1 may play a role in the stabilizing effect of poly(A) tail guanylation.

Mycb and Mych stimulate Müller glial cell reprogramming and proliferation in the uninjured and injured zebrafish retina.

In the injured zebrafish retina, Müller glial cells (MG) reprogram to adopt retinal stem cell properties and regenerate damaged neurons. The strongest zebrafish reprogramming factors might be good candidates for stimulating a similar regenerative response by mammalian MG. Myc proteins are potent reprogramming factors that can stimulate cellular plasticity in differentiated cells; however, their role in MG reprogramming and retina regeneration remains poorly explored. Here, we report that retinal injury stimulates mycb and mych expression and that, although both Mycb and Mych stimulate MG reprogramming and proliferation, only Mych enhances retinal neuron apoptosis. RNA-sequencing analysis of wild-type, mychmut and mycbmut fish revealed that Mycb and Mych regulate ∼40% and ∼16%, respectively, of the genes contributing to the regeneration-associated transcriptome of MG. Of these genes, those that are induced are biased towards regulation of ribosome biogenesis, protein synthesis, DNA synthesis, and cell division, which are the top cellular processes affected by retinal injury, suggesting that Mycb and Mych are potent MG reprogramming factors. Consistent with this, forced expression of either of these proteins is sufficient to stimulate MG proliferation in the uninjured retina.

Biosynthesis of Strained Amino Acids by a PLP‐Dependent Enzyme through Cryptic Halogenation

AbstractAmino acids (AAs) are modular building blocks which nature uses to synthesize both macromolecules, such as proteins, and small molecule natural products, such as alkaloids and non‐ribosomal peptides. While the 20 main proteinogenic AAs display relatively limited side chain diversity, a wide range of non‐canonical amino acids (ncAAs) exist that are not used by the ribosome for protein synthesis, but contain a broad array of structural features and functional groups. In this communication, we report the discovery of the biosynthetic pathway for a new ncAA, pazamine, which contains a cyclopropane ring formed in two steps. In the first step, a chlorine is added onto the C4 position of lysine by a radical halogenase, PazA. The cyclopropane ring is then formed in the next step by a pyridoxal‐5′‐phosphate‐dependent enzyme, PazB, via an SN2‐like attack at C4 to eliminate chloride. Genetic studies of this pathway in the native host, Pseudomonas azotoformans, show that pazamine potentially inhibits ethylene biosynthesis in growing plants based on alterations in the root phenotype of Arabidopsis thaliana seedlings. We further show that PazB can be utilized to make an alternative cyclobutane‐containing AA. These discoveries may lead to advances in biocatalytic production of specialty chemicals and agricultural biotechnology.

Biosynthesis of Strained Amino Acids by a PLP-Dependent Enzyme through Cryptic Halogenation.

Amino acids (AAs) are modular building blocks which nature uses to synthesize both macromolecules, such as proteins, and small molecule natural products, such as alkaloids and non-ribosomal peptides. While the 20 main proteinogenic AAs display relatively limited side chain diversity, a wide range of non-canonical amino acids (ncAAs) exist that are not used by the ribosome for protein synthesis, but contain a broad array of structural features and functional groups. In this communication, we report the discovery of the biosynthetic pathway for a new ncAA, pazamine, which contains a cyclopropane ring formed in two steps. In the first step, a chlorine is added onto the C4 position of lysine by a radical halogenase, PazA. The cyclopropane ring is then formed in the next step by a pyridoxal-5'-phosphate-dependent enzyme, PazB, via an SN2-like attack at C4 to eliminate chloride. Genetic studies of this pathway in the native host, Pseudomonas azotoformans, show that pazamine potentially inhibits ethylene biosynthesis in growing plants based on alterations in the root phenotype of Arabidopsis thaliana seedlings. We further show that PazB can be utilized to make an alternative cyclobutane-containing AA. These discoveries may lead to advances in biocatalytic production of specialty chemicals and agricultural biotechnology.

Implications of m5C modifications in ribosomal proteins on oxidative stress, metabolic reprogramming, and immune responses in patients with mid-to-late-stage head and neck squamous cell carcinoma: Insights from nanopore sequencing

BackgroundHead and Neck Squamous Cell Carcinoma (HNSCC) is a malignancy characterized by a high incidence and recurrence rate. 5-methylcytosine (m5C) RNA modification is a common alteration affecting cancer progression; however, how m5C operates within the tumor microenvironment of HNSCC remains to be elucidated. MethodsWe conducted Nanopore sequencing on 3 pairs of cancer and paracancerous tissues from mid- and late-stage HNSCC, obtaining 132 upregulated genes (transcriptomically upregulated, m5C elevated) and 129 downregulated genes (transcriptomically downregulated, m5C reduced). Subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed; a differential gene interaction network (PPI) was constructed, revealing the interactions of each gene with others in the network. Co-expression analysis was performed on the genes within the PPI, unveiling their expression and regulatory relationships. Through GSVA analysis, variations in related pathways under different states were identified. Furthermore, results of m5C in lncRNA were screened, followed by target gene prediction. ResultsSequencing results from the 3 pairs of mid- and late-stage HNSCC cancer and paracancerous tissues demonstrated that RPS27A, RPL8, and the lncRNAs including differentiation antagonizing nonprotein coding RNA (DANCR), DCST1 antisense RNA 1 (CCDC144NL-AS1), Growth Arrest-Specific Transcript 5 (GAS5), Nuclear Paraspeckle Assembly Transcript 1 (NEAT1), and Small Nucleolar RNA Host Gene 3 (SNHG3), etc., under m5Cregulation, have close connections with surrounding genes. The differentially m5Cmodified genes are primarily involved in ribosomal protein synthesis, oxidative stress response, metabolic reprogramming, immunity, and other life processes; pathways like mitochondrial protein import and photodynamic therapy induced unfolded protein response are upregulated in the tumor, while pathways, including the classic P53, are suppressed. Analysis on m5C-regulated long non-coding RNAs (lncRNAs) revealed tight associations with RPS27A and RPL8 as well. ConclusionOur study identifies the key factors and signaling pathways involving m5C in HNSCC. The findings suggest that ribosome-related genes might regulate ribosomal protein synthesis, oxidative stress response, metabolic reprogramming, and immune response through m5C RNA modification by means like hypoxia and ferroptosis, thereby playing a pivotal role in the onset and progression of HNSCC. Hence, attention should be paid to the role of ribosomes in HNSCC. These findings may facilitate the precision and individualized treatment of patients with mid- and late-stage HNSCC in clinical settings.

Abstract PR003: Mechanisms of therapeutic vulnerability of mismatch repair defective cancers to RNA polymerase I inhibitors

Abstract The goal of this study was to identify cancer types with synthetic vulnerability to RNA polymerase I (Pol I) inhibitors and to resolve mechanistic underpinnings of this vulnerability. Specific and selective Pol I inhibitors, pioneered by our group, block the transcription of rRNA, which is the rate-limiting step in ribosome biogenesis and protein synthesis. We conducted unbiased large-scale cancer cell line screens with the Pol I inhibitors and performed correlative analyses using DepMap and Achilles databases to identify cancer types and features that sensitize cancer cells to these inhibitors. The most sensitive lineages were colorectal, endometrial, esophageal, leukemia, myeloma, and ovarian. Microsatellite instable (MSI) cancer cell lines were highly sensitive and significantly more sensitive than microsatellite stable lines. Comparison of the cancer cell line responses to the Achilles gene essentiality database showed high correlation of the sensitivity to genetic knock-out of three Pol I subunits. This unbiased finding strongly supports identification of Pol I as the target. Correlative analyses for vulnerability identified two ribosomal proteins, RPL22 and RPL22L1, and MDM4, a p53 transcriptional repressor as the top candidates. Notably, RPL22K15Rfs hotspot mutation, the top-ranking genetic correlate, is present in up 70% MSI cancer cell lines and tumors. RPL22 and RPL22L1 are a synthetic lethal paralog pair. RPL22 is a negative regulator of RPL22L1 and consequently, haploinsufficiency or loss of RPL22 induces RPL22L1. RPL22L1 was the top-ranking expression correlate. Lastly, MDM4 was the top protein marker and is highly expressed in MSI cancers. These findings were validated using genetic and mechanistic analyses. We show that either genetic or chemical inhibition of the Pol I enzyme decreases the expression of RPL22L1 and MDM4 and alters their splicing. RPL22L1 is spliced to an alternative 3’ splice site in intron 2 leading to an early truncation. MDM4 exon 6 skipping converts the full-length p53 repressive form to a short NMD-targeted form. Mechanistically, by using GoldCLIP-seq, rMATS and splicing analyses, we show that RPL22 is a master regulator of splicing by binding to intronic sequences and splice sites of RPL22L1 and MDM4. Finally, we show the efficacy and tolerance of the Pol I inhibitors in xenograft and patient-derived MSI models expressing these markers. In summary, this synthetic vulnerability screen using new Pol I inhibitors uncovered a connection between Pol I transcription and cellular splicing programs and identified perturbation of this connection in MSI cancers. We conclude that RPL22 mutation could serve as tumor-agnostic biomarker for targeting cancers by Pol I inhibitors. Furthermore, this work identifies a new Pol I transcription-dependent ribosomal protein-governed pathway that converges on the regulation of p53. Citation Format: Marikki Laiho, Wenjun Fan, Hester Liu, Gregory C. Stachelek, Asma Begum, Catherine E. Davis, Tony Dorado, Pablo de Leon, Rajeshkumar N.V., James C. Barrow. Mechanisms of therapeutic vulnerability of mismatch repair defective cancers to RNA polymerase I inhibitors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr PR003.

Ribosomal dormancy at the nexus of ribosome homeostasis and protein synthesis.

Dormancy or hibernation is a non-proliferative state of cells with low metabolic activity and gene expression. Dormant cells sequester ribosomes in a translationally inactive state, called dormant/hibernating ribosomes. These dormant ribosomes are important for the preservation of ribosomes and translation shut-off. While recent studies attempted to elucidate their modes of formation, the regulation and roles of the diverse dormant ribosomal populations are still largely understudied. The mechanistic details of the formation of dormant ribosomes in stress and especially their disassembly during recovery remain elusive. In this review, we discuss the roles of dormant ribosomes and their potential regulatory mechanisms. Furthermore, we highlight the paradigms that need to be answered in the field of ribosomal dormancy.

Abstract PO5-06-11: RPL27A promotes the growth and metastasis of triple negative breast cancer by regulating Ribosome biogenesis

Abstract Background: Ribosome is a complex structure in cells for protein synthesis. It has been considered always have a constant composition. Recent studies have revealed that the Ribosome composition of tumor cells is heterogeneous. The Ribosome protein composition, post-translational modification, rRNA modification, and rRNA variants can lead to the existence of specialized "onco-ribosomes" in tumor cells, which regulates the rate of translation and protein synthesis, and can affect the occurrence and development of tumors significantly. The abnormal expression of multiple Ribosome proteins in triple-negative breast cancer may be the target of its diagnosis and treatment, also, an independent factor in prognosis evaluation. Methods: To learn the expression levels of Ribosome biogenesis related genes in normal breast cells and abnormal breast cells with different pathological subtypes of breast cancer, the following studies have been done: 1. Bioinformatics analysis on TCGA database and single-cell sequencing data; 2. IHC detection on TNBC tissue and adjacent tissues; 3. Determine the expression level of RPL27A in TNBC cells, and analyze the correlation between the expression of RPL27A and clinical information of patients. To explore the effect of RPL27A on Ribosome biogenesis, protein synthesis and other signaling pathways through Proteomics and molecular biology experiments have been designed and tested. The effects of RPL27A on malignant biological behaviors such as proliferation, migration, infiltration, and clonal formation of TNBC cells were studied through cell biology experiments and mouse experiments. Results: The analysis of TCGA database and single-cell sequencing data showed that the RPL27A was significantly increased in TNBC group. Immunohistochemical experiments showed that RPL27A was highly expressed in TNBC tissue and was impressively positively correlated with tumor size, lymph node metastasis, and bone metastasis. There was significantly negatively correlated with overall survival (OS) and disease free survival (DFS). It can be an independent factor for evaluating the prognosis of TNBC. By knocking out RPL27A in TNBC cell line, label free proteomics analysis showed that Ribosome biogenesis, protein synthesis, cell cycle regulation and other signaling pathways were obviously down regulated. Cell biology experiments have implied that the high expression of RPL27A in TNBC promotes cell proliferation, enhances cell migration, infiltration, and cloning ability, leads to more malignant biological behavior of tumor cells. Further research demonstrates that the high expression of RPL27A in TNBC significantly activated the EIF3C signal, cause changes in the composition of Ribosome in TNBC cells, promoted the formation of "onco-ribosomes", and distinctly increased the rate of protein synthesis. The study of PDX mice and Organoid showed that the knock out of RPL27A significantly reduced the growth rate and transfer ability of TNBC. Conclusion: The obvious over expressing of RPL27A in TNBC, which can significantly promote the growth and metastasis of tumor cells by influencing the Ribosome biogenesis process. Citation Format: Weipeng Zhao, Jingyi Zhang, Ye Zhu, Xin Yang, Xichuan Li. RPL27A promotes the growth and metastasis of triple negative breast cancer by regulating Ribosome biogenesis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-06-11.

AICAR confers prophylactic cardioprotection in doxorubicin-induced heart failure in rats

Doxorubicin (DOX) is a widely used chemotherapeutic agent that can cause serious cardiotoxic side effects, leading to heart failure (HF). Impaired mitochondrial function is thought to be key factor driving progression into HF. We have previously shown in a rat model of DOX-HF that heart failure with reduced ejection fraction correlates with mitochondrial loss and dysfunction. Adenosine monophosphate-dependent kinase (AMPK) is a cellular energy sensor, regulating mitochondrial biogenesis and energy metabolism, including fatty acid oxidation. We hypothesised that AMPK activation could restore mitochondrial function and therefore be a novel cardioprotective strategy for the prevention of DOX-HF. Consequently, we set out to assess whether 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR), an activator of AMPK, could prevent cardiac functional decline in this chronic intravenous rat model of DOX-HF. In line with our hypothesis, AICAR improved cardiac systolic function. AICAR furthermore improved cardiac mitochondrial fatty acid oxidation, independent of mitochondrial number, and in the absence of observable AMPK-activation. In addition, we found that AICAR prevented loss of myocardial mass. RNAseq analysis showed that this may be driven by normalisation of pathways associated with ribosome function and protein synthesis, which are impaired in DOX-treated rat hearts. AICAR furthermore prevented dyslipidemia and excessive body-weight loss in DOX-treated rats, which may contribute to preservation of myocardial mass. Though it is unclear whether AICAR exerted its cardioprotective effect through cardiac or extra-cardiac AMPK-activation or via an AMPK-independent effect, these results show promise for the use of AICAR as a cardioprotective agent in DOX-HF to both preserve cardiac function and mass.

Critical cis-parameters influence STructure assisted RNA translation (START) initiation on non-AUG codons in eukaryotes.

In eukaryotes, translation initiation is a highly regulated process, which combines cis-regulatory sequences located on the messenger RNA along with trans-acting factors like eukaryotic initiation factors (eIF). One critical step of translation initiation is the start codon recognition by the scanning 43S particle, which leads to ribosome assembly and protein synthesis. In this study, we investigated the involvement of secondary structures downstream the initiation codon in the so-called START (STructure-Assisted RNA translation) mechanism on AUG and non-AUG translation initiation. The results demonstrate that downstream secondary structures can efficiently promote non-AUG translation initiation if they are sufficiently stable to stall a scanning 43S particle and if they are located at an optimal distance from non-AUG codons to stabilize the codon-anticodon base pairing in the P site. The required stability of the downstream structure for efficient translation initiation varies in distinct cell types. We extended this study to genome-wide analysis of functionally characterized alternative translation initiation sites in Homo sapiens. This analysis revealed that about 25% of these sites have an optimally located downstream secondary structure of adequate stability which could elicit START, regardless of the start codon. We validated the impact of these structures on translation initiation for several selected uORFs.

Pan-cancer analysis of ARFs family and ARF5 promoted the progression of hepatocellular carcinoma

BackgroundARF family proteins are a kind of small GTPases, which are involved in regulating a variety of basic functions of cells. In recent years, the role and molecular regulatory mechanisms of ARFs in tumor progression have received increasing attention, and research reports on most of their family members are increasing. However, research on the clinical and pathological relevance of ARF5 in cancer, especially in hepatocellular carcinoma, still needs to be improved. MethodsRNA-seq data in the Cancer Genome Atlas (TCGA) and genome tissue expression (GTEx) databases were used to analyze the expression and pathological data of ARFs family in Pan-cancer. Kaplan-Meier and Cox regression were used for prognostic analysis of ARF5 and Pan-cancer. Combined with ImmuCellAI database and TIMER2 database, the relationship between ARF5 expression and immune cell tumor infiltration in hepatocellular carcinoma (HCC) was analyzed. WGCNA is used to construct the co-expression gene network related to ARF5 expression in HCC and screen important modules and central genes. GO and KEGG path enrichment analysis were carried out for the genes in the modules with clinical significance. GSEA analysis was performed to take into account the role of genes with small differences. Finally, ceRNA network analysis was used to explore the molecular mechanism of miRNAs and lncRNAs regulating ARF5 expression. ResultsARFs family (ARF1, ARF3, ARF4, ARF5, ARF6) are generally highly expressed in Pan-cancer. ARF5 is significantly highly expressed in 29 cancers, and the high expression of ARF5 in HCC patients is significantly negatively correlated with OS, DFI, PFI and DSS, which may lead to cancer deterioration by participating in tumor immune infiltration of HCC. Through WGCNA analysis, the expression of ARF5 in HCC may be involved in many cellular processes that consume a lot of energy, such as ribosome formation, RNA and protein synthesis and lipids, as well as COVID-19, nonalcoholic fatty liver, neurodegenerative diseases and other disease pathways. ConclusionARFs, especially ARF5, are overexpressed in many human tumors. This study shows for the first time that ARF5 is significantly correlated with the poor prognosis of HCC patients, which may play a role as an oncogene, suggesting that ARF5 has the potential as a biomarker for the diagnosis and treatment of HCC.

Sirolimus experience in adult patients with vascular malformations.

Sirolimus, a mammalian target of rapamycin inhibitor, inhibits cell growth and proliferation by controlling ribosome biogenesis and protein synthesis in vascular anomalies and cancers. However, most sirolimus studies on vascular anomalies were conducted in the pediatric population, with limited data in adults. In this study, we assessed the effectiveness and safety of sirolimus in adult patients with vascular malformation, a subtype of vascular anomaly. We conducted a retrospective analysis of adult vascular malformation patients aged over 16, treated at Hacettepe University Cancer Institute from January 2013 to September 2022. Patient demographics and clinical characteristics were recorded. The primary outcome was the efficacy of sirolimus evaluated by response and disease control rates. The disease control rate was defined as the cumulative percentage of complete or partial responses, along with stable disease. The secondary endpoint was toxicity and safety. 38 patients with a median age of 21 (IQR: 18-33) were recruited. Prior to sirolimus treatment, 57.9% of patients had undergone other therapeutic interventions, predominantly sclerotherapy and surgery. The median follow-up time during sirolimus treatment was 18.5 (IQR: 11.3-74.5) months. The disease control rate was 92.1% (35/38). Head-neck localization was associated with better response rates (p = .001). Sirolimus was generally well tolerated and grade 1 or 2 oral mucositis (n = 4) and skin rash (n = 3) were the most common side effects. In this study, we found sirolimus was efficacious and well tolerated in adult patients with vascular malformation.

Prognostic gene biomarkers for c-Src inhibitor Si162 sensitivity in melanoma cells.

Early detection and treatment are crucial in combating malignant melanoma. Src is an important therapeutic target in melanoma due to its association with cancer progression. However, developing effective Src-targeting drugs remains challenging and personalized medicine relies on biomarkers and targeted therapies for precise and effective treatment. This study focuses on Si162, a newly synthesized c-Src inhibitor, to identify reliable biomarkers for predicting Si162 sensitivity and explore associated biological characteristics and pathways in melanoma cells. Primary melanoma cells (M1, M21, M24, M84, M133, M307, and M2025) were obtained from patients diagnosed with melanoma. Si162 cytotoxicity tests were performed using luminescent adenosine triphosphate detection and the half-maximal inhibitory concentration (IC50) values were calculated. Gene expression profiles were analyzed using microarray-based gene expression data. Differentially expressed genes between the resistant and sensitive groups were identified using Pearson correlation analysis. Gene coexpression, interactions, and pathways were investigated through clustering, network, and pathway analyses. Biological functions were examined using the Database for Annotation, Visualization, and Integrated Discovery. Molecular pathways associated with different responses to Si162 were identified using gene set enrichment analysis. The gene expressions were validated using reverse transcription-quantitative polymerase chain reaction. The cells revealed significant differences in response to Si162 based on the IC50 values (p < 0.05). A total of 36 differentially expressed genes associated with Si162 susceptibility were identified. Distinct expression patterns between the sensitive and resistant groups were observed in 9 genes (LRBA, MGMT, CAND1, ADD1, SETD2, CNTN6, FGF18, C18orf25, and RPL13). Coexpression among the differentially expressed genes was highlighted, and 9 genes associated with molecular pathways, including EMT, transforming growth factor-beta (TGF-β) signaling, and ribosomal protein synthesis, between groups. Genes involved in dysregulated immune response were observed in the resistant group. The involvement of 5 genes (ADD1, CNTN6, FGF18, C18orf25, and RPL13) in Si162 resistance was confirmed through qRT-PCR validation. These findings contribute to our understanding of the underlying biological differences among melanoma cells and suggest potential biomarkers and pathways associated with Si162 response and resistance.

Proteome-Wide Analysis of Lysine 2-Hydroxyisobutyrylation in Aspergillus fumigatus

Aspergillus fumigatus is the significant causative agent in cases of invasive aspergillosis, leading to a high mortality rate in immunocompromised patients. A comprehensive understanding of its growth patterns and metabolic processes within the host is a critical prerequisite for the development of effective antifungal strategies. Lysine 2-hydroxyisobutyrylation (Khib) is a highly conserved protein posttranslational modifications (PTM) found in various organisms. In this study, we investigate the biological impact of Khib in A. fumigatus. Using a combination of antibody enrichment with the conventional LC–MS/MS method, the pattern of Khib-modification in proteins and their respective sites were analyzed in a wild type strain of A. fumigatus. Our findings revealed 3494 Khib-modified proteins with a total of 18,091 modified sites in this strain. Functional enrichment analysis indicated that these Khib-modified proteins participate in a diverse range of cellular functions, spanning various subcellular locations such as ribosome biosynthesis, protein synthesis and nucleocytoplasmic transport. Notably, when compared with other reported eukaryotes, A. fumigatus exhibited consistently higher numbers of Khib-modified proteins, suggesting the potential significance of this modification in this organism. An interesting observation is the prevalence of Khib modifications in most enzymes involved in the ergosterol synthesis pathway. The insights gathered from this study provide new avenue for studying PTM-associated mechanisms in fungal growth and offer potential implication for antifungal drug development.