Isocitrate dehydrogenase wt and IDHmut adult-type diffuse gliomas display distinct alterations in ribosome biogenesis and 2'O-methylation of ribosomal RNA.

BACKGROUND: High-grade adult-type diffuse gliomas (HGGs) constitute a heterogeneous group of aggressive tumors that are mostly incurable. Recent advances highlighting the contribution of ribosomes to cancer development have offered new clinical perspectives. Here, we uncovered that isocitrate dehydrogenase (IDH)wt and IDHmut HGGs display distinct alterations of ribosome biology, in terms of rRNA epitranscriptomics and ribosome biogenesis, which could constitute novel hallmarks that can be exploited for the management of these pathologies. METHODS: We analyzed (1) the ribosomal RNA 2'O-ribose methylation (rRNA 2'Ome) using RiboMethSeq and in-house developed bioinformatics tools (https://github.com/RibosomeCRCL/ribomethseq-nfandrRMSAnalyzer) on 3 independent cohorts compiling 71 HGGs (IDHwt n = 30, IDHmut n = 41) and 9 non-neoplastic samples, (2) the expression of ribosome biogenesis factors using medium throughput RT-qPCR as a readout of ribosome biogenesis, and (3) the sensitivity of 5 HGG cell lines to RNA Pol I inhibitors (CX5461, BMH-21). RESULTS: Unsupervised analysis demonstrated that HGGs could be distinguished based on their rRNA 2'Ome epitranscriptomic profile, with IDHwt glioblastomas displaying the most significant alterations of rRNA 2'Ome at specific sites. In contrast, IDHmut HGGs are largely characterized by an overexpression of ribosome biogenesis factors compared to non-neoplastic tissues or IDHwt glioblastomas. Finally, IDHmut HGG-derived spheroids display higher cytotoxicity to CX5461 than IDHwt glioblastoma, while all HGG spheroids display a similar cytotoxicity to BMH-21. CONCLUSIONS: In HGGs, IDH mutational status is associated with specific alterations of the ribosome biology and with distinct sensitivities to RNA Pol I inhibitors.

Ribosome biogenesis-based predictive biomarkers in endocrine therapy (Anastrozole) combined with mTOR inhibitor (Vistusertib) in endometrial cancer: translational study from the VICTORIA trial in collaboration with the GINECO group.

Resistance of advanced hormone-dependent endometrial carcinoma to endocrine therapy remains a worldwide clinical issue. We recently reported that the combination of Vistusertib (V, mTOR inhibitor) and Anastrozole (A, aromatase inhibitor) improves the progression-free rate compared to Anastrozole alone. However, a better patient selection based on biomarkers would improve patient outcome. We evaluate for the first time the usage of ribosome biogenesis (RiBi) factors as a source of innovative markers. Using 47 FFPE tumours (A n = 18; V + A n = 29), 32 blood samples (A n = 13; V + A n = 19) and 30 samples of total RNAs (A n = 12; V + A n = 18) from the VICTORIA clinical trial, we observed an association between RiBi-associated markers and drug activity or prediction of treatment response. NOP10 and NHP2 mRNA levels were significantly higher in non-responders compared to responders in the Vistusertib + Anastrozole arm (P = 0.0194 and P = 0.0002 respectively; i.e. 8 weeks progression-free survival as endpoint). This study provides RiBi-based markers relevant for a better selection of patients with advanced endometrial carcinoma by predicting the response of endocrine therapy combined with mTOR inhibitor.

snoDB 2.0: an enhanced interactive database, specializing in human snoRNAs.

snoDB is an interactive database of human small nucleolar RNAs (snoRNAs) that includes up-to-date information on snoRNA features, genomic location, conservation, host gene, snoRNA-RNA targets and snoRNA abundance and provides links to other resources. In the second edition of this database (snoDB 2.0), we added an entirely new section on ribosomal RNA (rRNA) chemical modifications guided by snoRNAs with easy navigation between the different rRNA versions used in the literature and experimentally measured levels of modification. We also included new layers of information, including snoRNA motifs, secondary structure prediction, snoRNA-protein interactions, copy annotations and low structure bias expression data in a wide panel of tissues and cell lines to bolster functional probing of snoRNA biology. Version 2.0 features updated identifiers, more links to external resources and duplicate entry resolution. As a result, snoDB 2.0, which is freely available at https://bioinfo-scottgroup.med.usherbrooke.ca/snoDB/, represents a one-stop shop for snoRNA features, rRNA modification targets, functional impact and potential regulators.

Safety and Efficacy of the mTOR Inhibitor, Vistusertib, Combined With Anastrozole in Patients With Hormone Receptor-Positive Recurrent or Metastatic Endometrial Cancer: The VICTORIA Multicenter, Open-label, Phase 1/2 Randomized Clinical Trial.

Importance: Endometrial cancer is often hormone-dependent and treated with aromatase inhibitors. The PI3K-AKT-mTOR pathway deregulation observed in endometrial cancer drives hormonal resistance, thus supporting the rationale of combining mTOR inhibitor with endocrine therapy. Objective: To evaluate the safety and efficacy of vistusertib in combination with anastrozole in the treatment of women with hormone receptor-positive recurrent or metastatic endometrial cancer. Design, Settings, and Participants: The VICTORIA study was a multicenter, open-label, randomized clinical trial that accrued 75 patients with hormone receptor-positive recurrent or metastatic endometrial cancer from 12 cancer centers in France in April 2016 to October 2019. After a safety run-in period, a Simon 2-stage design was used. Data analyses were performed from December 11, 2020, to March 11, 2021. Interventions: Patients were randomized in a 2:1 ratio to oral vistusertib (125 mg twice daily 2 days per week) and oral anastrozole (1 mg daily) in the combination vistusertib with anastrozole arm (V+A arm) or oral anastrozole alone (A arm). Main Outcomes and Measures: The primary end point was serious adverse events for the safety run-in period and progression-free rate at 8 weeks (8wk-PFR)-assessed with a blinded independent central review in phase 2. The secondary end points were objective response rate, duration of response, progression-free survival (PFS), overall survival, and incidence of adverse events. Results: Of the 75 patients who were randomized, 73 (median [range] age, 69.5 [37-88] y; all female) were treated: V+A arm, 49 patients; A arm, 24 patients. In the V+A arm, the 8wk-PFR was 67.3% (unilateral 95% CI, 54.7%) and in the A arm, 39.1% (unilateral 95% CI, 22.2%). No significant serious adverse events were reported during the safety run-in period (n = 6 in V+A arm). The overall response rate was 24.5% (95% CI, 13.3%-38.9%) in the V+A arm vs 17.4% (95% CI, 5.0%-38.8%) in the A arm. With a median follow-up of 27.7 months, median PFS was 5.2 (95% CI, 3.4-8.9) in the V+A arm and 1.9 (95% CI, 1.6-8.9) months in the A arm. Fatigue, lymphopenia, hyperglycemia, and diarrhea were the most common (grade ≥2) adverse events associated with vistusertib. Conclusions and Relevance: This multicenter, open-label, phase 1/2 randomized clinical trial demonstrated that adding vistusertib to anastrozole improved 8wk-PFR, overall response rate, and PFS for patients with endometrial cancer and had manageable adverse events. Identification of molecular subgroups would allow for more precise selection of patients who may be most likely to experience favorable outcomes. Trial Registration: ClinicalTrials.gov Identifier: NCT02730923.

Low level of Fibrillarin, a ribosome biogenesis factor, is a new independent marker of poor outcome in breast cancer.

BACKGROUND: A current critical need remains in the identification of prognostic and predictive markers in early breast cancer. It appears that a distinctive trait of cancer cells is their addiction to hyperactivation of ribosome biogenesis. Thus, ribosome biogenesis might be an innovative source of biomarkers that remains to be evaluated. METHODS: Here, fibrillarin (FBL) was used as a surrogate marker of ribosome biogenesis due to its essential role in the early steps of ribosome biogenesis and its association with poor prognosis in breast cancer when overexpressed. Using 3,275 non-metastatic primary breast tumors, we analysed FBL mRNA expression levels and protein nucleolar organisation. Usage of TCGA dataset allowed transcriptomic comparison between the different FBL expression levels-related breast tumours. RESULTS: We unexpectedly discovered that in addition to breast tumours expressing high level of FBL, about 10% of the breast tumors express low level of FBL. A correlation between low FBL mRNA level and lack of FBL detection at protein level using immunohistochemistry was observed. Interestingly, multivariate analyses revealed that these low FBL tumors displayed poor outcome compared to current clinical gold standards. Transcriptomic data revealed that FBL expression is proportionally associated with distinct amount of ribosomes, low FBL level being associated with low amount of ribosomes. Moreover, the molecular programs supported by low and high FBL expressing tumors were distinct. CONCLUSION: Altogether, we identified FBL as a powerful ribosome biogenesis-related independent marker of breast cancer outcome. Surprisingly we unveil a dual association of the ribosome biogenesis FBL factor with prognosis. These data suggest that hyper- but also hypo-activation of ribosome biogenesis are molecular traits of distinct tumors.

Heterogeneity and dynamic of EMT through the plasticity of ribosome and mRNA translation.

Growing evidence exposes translation and its translational machinery as key players in establishing and maintaining physiological and pathological biological processes. Examining translation may not only provide new biological insight but also identify novel innovative therapeutic targets in several fields of biology, including that of epithelial-to-mesenchymal transition (EMT). EMT is currently considered as a dynamic and reversible transdifferentiation process sustaining the transition from an epithelial to mesenchymal phenotype, known to be mainly driven by transcriptional reprogramming. However, it seems that the characterization of EMT plasticity is challenging, relying exclusively on transcriptomic and epigenetic approaches. Indeed, heterogeneity in EMT programs was reported to depend on the biological context. Here, by reviewing the involvement of translational control, translational machinery and ribosome biogenesis characterizing the different types of EMT, from embryonic and adult physiological to pathological contexts, we discuss the added value of integrating translational control and its machinery to depict the heterogeneity and dynamics of EMT programs.

Alteration of ribosome function upon 5-fluorouracil treatment favors cancer cell drug-tolerance.

Mechanisms of drug-tolerance remain poorly understood and have been linked to genomic but also to non-genomic processes. 5-fluorouracil (5-FU), the most widely used chemotherapy in oncology is associated with resistance. While prescribed as an inhibitor of DNA replication, 5-FU alters all RNA pathways. Here, we show that 5-FU treatment leads to the production of fluorinated ribosomes exhibiting altered translational activities. 5-FU is incorporated into ribosomal RNAs of mature ribosomes in cancer cell lines, colorectal xenografts, and human tumors. Fluorinated ribosomes appear to be functional, yet, they display a selective translational activity towards mRNAs depending on the nature of their 5'-untranslated region. As a result, we find that sustained translation of IGF-1R mRNA, which encodes one of the most potent cell survival effectors, promotes the survival of 5-FU-treated colorectal cancer cells. Altogether, our results demonstrate that "man-made" fluorinated ribosomes favor the drug-tolerant cellular phenotype by promoting translation of survival genes.

MiRNAs and snoRNAs in Bone Metastasis: Functional Roles and Clinical Potential.

Bone is a frequent site of metastasis. Bone metastasis is associated with a short-term prognosis in cancer patients, and current treatments aim to slow its growth, but are rarely curative. Thus, revealing molecular mechanisms that explain why metastatic cells are attracted to the bone micro-environment, and how they successfully settle in the bone marrow-taking advantage over bone resident cells-and grow into macro-metastasis, is essential to propose new therapeutic approaches. MicroRNAs and snoRNAs are two classes of small non-coding RNAs that post-transcriptionally regulate gene expression. Recently, microRNAs and snoRNAs have been pointed out as important players in bone metastasis by (i) preparing the pre-metastatic niche, directly and indirectly affecting the activities of osteoclasts and osteoblasts, (ii) promoting metastatic properties within cancer cells, and (iii) acting as mediators within cells to support cancer cell growth in bone. This review aims to highlight the importance of microRNAs and snoRNAs in metastasis, specifically in bone, and how their roles can be linked together. We then discuss how microRNAs and snoRNAs are secreted by cancer cells and be found as extracellular vesicle cargo. Finally, we provide evidence of how microRNAs and snoRNAs can be potential therapeutic targets, at least in pre-clinical settings, and how their detection in liquid biopsies can be a useful diagnostic and/or prognostic biomarker to predict the risk of relapse in cancer patients.

A novel view on an old drug, 5-fluorouracil: an unexpected RNA modifier with intriguing impact on cancer cell fate.

5-Fluorouracil (5-FU) is a chemotherapeutic drug widely used to treat patients with solid tumours, such as colorectal and pancreatic cancers. Colorectal cancer (CRC) is the second leading cause of cancer-related death and half of patients experience tumour recurrence. Used for over 60 years, 5-FU was long thought to exert its cytotoxic effects by altering DNA metabolism. However, 5-FU mode of action is more complex than previously anticipated since 5-FU is an extrinsic source of RNA modifications through its ability to be incorporated into most classes of RNA. In particular, a recent report highlighted that, by its integration into the most abundant RNA, namely ribosomal RNA (rRNA), 5-FU creates fluorinated active ribosomes and induces translational reprogramming. Here, we review the historical knowledge of 5-FU mode of action and discuss progress in the field of 5-FU-induced RNA modifications. The case of rRNA, the essential component of ribosome and translational activity, and the plasticity of which was recently associated with cancer, is highlighted. We propose that translational reprogramming, induced by 5-FU integration in ribosomes, contributes to 5-FU-driven cell plasticity and ultimately to relapse.

2'O-Ribose Methylation of Ribosomal RNAs: Natural Diversity in Living Organisms, Biological Processes, and Diseases.

Recent findings suggest that ribosomes, the translational machineries, can display a distinct composition depending on physio-pathological contexts. Thanks to outstanding technological breakthroughs, many studies have reported that variations of rRNA modifications, and more particularly the most abundant rRNA chemical modification, the rRNA 2'O-ribose methylation (2'Ome), intrinsically occur in many organisms. In the last 5 years, accumulating reports have illustrated that rRNA 2'Ome varies in human cell lines but also in living organisms (yeast, plant, zebrafish, mouse, human) during development and diseases. These rRNA 2'Ome variations occur either within a single cell line, organ, or patient's sample (i.e., intra-variability) or between at least two biological conditions (i.e., inter-variability). Thus, the ribosomes can tolerate the absence of 2'Ome at some specific positions. These observations question whether variations in rRNA 2'Ome could provide ribosomes with particular translational regulatory activities and functional specializations. Here, we compile recent studies supporting the heterogeneity of ribosome composition at rRNA 2'Ome level and provide an overview of the natural diversity in rRNA 2'Ome that has been reported up to now throughout the kingdom of life. Moreover, we discuss the little evidence that suggests that variations of rRNA 2'Ome can effectively impact the ribosome activity and contribute to the etiology of some human diseases.

Erratum: Ribosomal RNA 2' O-methylation as a novel layer of inter-tumour heterogeneity in breast cancer.

[This corrects the article DOI: 10.1093/narcan/zcaa036.].

Ribosome Biogenesis Alterations in Colorectal Cancer.

Many studies have focused on understanding the regulation and functions of aberrant protein synthesis in colorectal cancer (CRC), leaving the ribosome, its main effector, relatively underappreciated in CRC. The production of functional ribosomes is initiated in the nucleolus, requires coordinated ribosomal RNA (rRNA) processing and ribosomal protein (RP) assembly, and is frequently hyperactivated to support the needs in protein synthesis essential to withstand unremitting cancer cell growth. This elevated ribosome production in cancer cells includes a strong alteration of ribosome biogenesis homeostasis that represents one of the hallmarks of cancer cells. None of the ribosome production steps escape this cancer-specific dysregulation. This review summarizes the early and late steps of ribosome biogenesis dysregulations described in CRC cell lines, intestinal organoids, CRC stem cells and mouse models, and their possible clinical implications. We highlight how this cancer-related ribosome biogenesis, both at quantitative and qualitative levels, can lead to the synthesis of ribosomes favoring the translation of mRNAs encoding hyperproliferative and survival factors. We also discuss whether cancer-related ribosome biogenesis is a mere consequence of cancer progression or is a causal factor in CRC, and how altered ribosome biogenesis pathways can represent effective targets to kill CRC cells. The association between exacerbated CRC cell growth and alteration of specific steps of ribosome biogenesis is highlighted as a key driver of tumorigenesis, providing promising perspectives for the implementation of predictive biomarkers and the development of new therapeutic drugs.

Uncovering the Translational Regulatory Activity of the Tumor Suppressor BRCA1.

BRCA1 inactivation is a hallmark of familial breast cancer, often associated with aggressive triple negative breast cancers. BRCA1 is a tumor suppressor with known functions in DNA repair, transcription regulation, cell cycle control, and apoptosis. In the present study, we demonstrate that BRCA1 is also a translational regulator. We previously showed that BRCA1 was implicated in translation regulation. Here, we asked whether translational control could be a novel function of BRCA1 that contributes to its tumor suppressive activity. A combination of RNA-binding protein immunoprecipitation, microarray analysis, and polysome profiling, was used to identify the mRNAs that were specifically deregulated under BRCA1 deficiency. Western blot analysis allowed us to confirm at the protein level the deregulated translation of a subset of mRNAs. A unique and dedicated cohort of patients with documented germ-line BRCA1 pathogenic variant statues was set up, and tissue microarrays with the biopsies of these patients were constructed and analyzed by immunohistochemistry for their content in each candidate protein. Here, we show that BRCA1 translationally regulates a subset of mRNAs with which it associates. These mRNAs code for proteins involved in major programs in cancer. Accordingly, the level of these key proteins is correlated with BRCA1 status in breast cancer cell lines and in patient breast tumors. ADAT2, one of these key proteins, is proposed as a predictive biomarker of efficacy of treatments recently recommended to patients with BRCA1 deficiency. This study proposes that translational control may represent a novel molecular mechanism with potential clinical impact through which BRCA1 is a tumor suppressor.

snoRNAs Offer Novel Insight and Promising Perspectives for Lung Cancer Understanding and Management.

Small nucleolar RNAs (snoRNAs) are non-coding RNAs localized in the nucleolus, where they participate in the cleavage and chemical modification of ribosomal RNAs. Their biogenesis and molecular functions have been extensively studied since their identification in the 1960s. However, their role in cancer has only recently started to emerge. In lung cancer, efforts to profile snoRNA expression have enabled the definition of snoRNA-related signatures, not only in tissues but also in biological fluids, exposing these small RNAs as potential non-invasive biomarkers. Moreover, snoRNAs appear to be essential actors of lung cancer onset and dissemination. They affect diverse cellular functions, from regulation of the cell proliferation/death balance to promotion of cancer cell plasticity. snoRNAs display both oncogenic and tumor suppressive activities that are pivotal in lung cancer tumorigenesis and progression. Altogether, we review how further insight into snoRNAs may improve our understanding of basic lung cancer biology and the development of innovative diagnostic tools and therapies.

Iron and hepcidin mediate human colorectal cancer cell growth.

High dietary iron intake is a risk factor for the development of colorectal cancer. However, how iron subsequently impacts the proliferation of colorectal cancer cells remains unclear. This study determined the expression of six iron regulatory genes in twenty-one human colorectal cancer (CRC) biopsies and matched normal colonic tissue. The results show that only hepcidin and ferritin heavy chain expression were increased in CRC biopsies as compared to matched normal tissues. Four established human CRC cell lines, HT-29, HCT-116, SW-620 and SW-480 were subsequently examined for their growth in response to increasing concentrations of iron, and iron depletion. Real time cell growth assay showed a significant inhibitory effect of acute iron loading in HCT-116 cells (IC50 = 258.25 µM at 72 h), and no significant effects in other cell types. However, ten week treatment with iron significantly reduced HT-29 and SW-620 cell growth, whereas no effect was seen in HCT-116 and SW-480 cells. Intracellular labile iron depletion induced the complete growth arrest and detachment of all of the CRC cell types except for the SW-620 cell line which was not affected in its growth. Treatment of starved CRC cells with hepcidin, the major regulator of iron metabolism, induced a significant stimulation of HT-29 cell growth but did not affect the growth of the other cell types. Collectively these results show that iron is central to CRC cell growth in a manner that is not identical between acute and chronic loading, and that is specific to the CRC cell type.

Analysis of the rRNA methylation complex components in pediatric B-cell precursor acute lymphoblastic leukemia: A pilot study.

BACKGROUND: Dysregulation of ribosome biogenesis and alteration of ribosome composition, including alteration in ribosomal RNA (rRNA) 2'-O-ribose methylation, can play a role in malignant transformation and cancer progression. Several studies recently reported that components of the rRNA methylation complex are associated with leukemogenesis. However, no study ever investigated the alteration of ribosome biogenesis factors in the most common pediatric malignancy - B-cell precursor acute lymphoblastic leukemia (BCP-ALL). OBJECTIVES: The objective of this study was to examine the expression of factors building the rRNA methylation complex, either the protein components (1 methyl-transferase (FBL), NOP56, NOP58, NHP2L1) or some RNA components (box C/D snoRNAs: SNORD35B, SNORD65, SNORD46, SNORD50A, SNORD38B), as well as CMYC, and nucleolin (NCL) - a protein involved in rRNA synthesis. Clinical effects in children with BCP-ALL were also investigated. MATERIAL AND METHODS: The factors involved in ribosome biogenesis were studied in 28 children with BCP-ALL with the use of real-time polymerase chain reaction (RT-PCR) using the BioMark HD System (Fluidigm, San Francisco, USA) in cDNA prepared from the bone marrow samples collected at diagnosis. RESULTS: Strong correlations were observed between NOP56, NOP58 and NCL, and multiple weaker correlations were observed in the box C/D snoRNA category, and between box C/D snoRNA and transcripts coding proteins of the rRNA methylation complex. The expression of analyzed transcripts did not correlate with the initial white blood cells count (WBC) or with bone marrow blast percentage. Ribosome biogenesis upregulation with overexpression of FBL and NOP56, and CMYC was found in patients who subsequently relapsed and the upregulation signature was not associated with known risk predictors. CONCLUSIONS: This is the first report on the clinical aspect of ribosome biogenesis in pediatric BCP-ALL, and it shows that overexpression of CMYC and C/D box nucleoproteins FBL and NOP56 is an antecedent event in patients who subsequently relapse. The dysregulation pattern is different from the previous reports in acute myeloid leukemia (AML), suggesting that dysregulation of ribosome biogenesis is specific for BCP-ALL.

Ribosomal RNA 2'O-methylation as a novel layer of inter-tumour heterogeneity in breast cancer.

Recent epitranscriptomics studies unravelled that ribosomal RNA (rRNA) 2'O-methylation is an additional layer of gene expression regulation highlighting the ribosome as a novel actor of translation control. However, this major finding lies on evidences coming mainly, if not exclusively, from cellular models. Using the innovative next-generation RiboMeth-seq technology, we established the first rRNA 2'O-methylation landscape in 195 primary human breast tumours. We uncovered the existence of compulsory/stable sites, which show limited inter-patient variability in their 2'O-methylation level, which map on functionally important sites of the human ribosome structure and which are surrounded by variable sites found from the second nucleotide layers. Our data demonstrate that some positions within the rRNA molecules can tolerate absence of 2'O-methylation in tumoral and healthy tissues. We also reveal that rRNA 2'O-methylation exhibits intra- and inter-patient variability in breast tumours. Its level is indeed differentially associated with breast cancer subtype and tumour grade. Altogether, our rRNA 2'O-methylation profiling of a large-scale human sample collection provides the first compelling evidence that ribosome variability occurs in humans and suggests that rRNA 2'O-methylation might represent a relevant element of tumour biology useful in clinic. This novel variability at molecular level offers an additional layer to capture the cancer heterogeneity and associates with specific features of tumour biology thus offering a novel targetable molecular signature in cancer.

Emerging Role of Eukaryote Ribosomes in Translational Control.

Translation is one of the final steps that regulate gene expression. The ribosome is the effector of translation through to its role in mRNA decoding and protein synthesis. Many mechanisms have been extensively described accounting for translational regulation. However it emerged only recently that ribosomes themselves could contribute to this regulation. Indeed, though it is well-known that the translational efficiency of the cell is linked to ribosome abundance, studies recently demonstrated that the composition of the ribosome could alter translation of specific mRNAs. Evidences suggest that according to the status, environment, development, or pathological conditions, cells produce different populations of ribosomes which differ in their ribosomal protein and/or RNA composition. Those observations gave rise to the concept of "specialized ribosomes", which proposes that a unique ribosome composition determines the translational activity of this ribosome. The current review will present how technological advances have participated in the emergence of this concept, and to which extent the literature sustains this concept today.