RNA Subtypes Research Articles

Mass Spectrometry-Based Pipeline for Identifying RNA Modifications Involved in a Functional Process: Application to Cancer Cell Adaptation.

Cancer onset and progression are known to be regulated by genetic and epigenetic events, including RNA modifications (a.k.a. epitranscriptomics). So far, more than 150 chemical modifications have been described in all RNA subtypes, including messenger, ribosomal, and transfer RNAs. RNA modifications and their regulators are known to be implicated in all steps of post-transcriptional regulation. The dysregulation of this complex yet delicate balance can contribute to disease evolution, particularly in the context of carcinogenesis, where cells are subjected to various stresses. We sought to discover RNA modifications involved in cancer cell adaptation to inhospitable environments, a peculiar feature of cancer stem cells (CSCs). We were particularly interested in the RNA marks that help the adaptation of cancer cells to suspension culture, which is often used as a surrogate to evaluate the tumorigenic potential. For this purpose, we designed an experimental pipeline consisting of four steps: (1) cell culture in different growth conditions to favor CSC survival; (2) simultaneous RNA subtype (mRNA, rRNA, tRNA) enrichment and RNA hydrolysis; (3) the multiplex analysis of nucleosides by LC-MS/MS followed by statistical/bioinformatic analysis; and (4) the functional validation of identified RNA marks. This study demonstrates that the RNA modification landscape evolves along with the cancer cell phenotype under growth constraints. Remarkably, we discovered a short epitranscriptomic signature, conserved across colorectal cancer cell lines and associated with enrichment in CSCs. Functional tests confirmed the importance of selected marks in the process of adaptation to suspension culture, confirming the validity of our approach and opening up interesting prospects in the field.

Multiplexed RNA profiling by regenerative catalysis enables blood-based subtyping of brain tumors

Current technologies to subtype glioblastoma (GBM), the most lethal brain tumor, require highly invasive brain biopsies. Here, we develop a dedicated analytical platform to achieve direct and multiplexed profiling of circulating RNAs in extracellular vesicles for blood-based GBM characterization. The technology, termed ‘enzyme ZIF-8 complexes for regenerative and catalytic digital detection of RNA’ (EZ-READ), leverages an RNA-responsive transducer to regeneratively convert and catalytically enhance signals from rare RNA targets. Each transducer comprises hybrid complexes – protein enzymes encapsulated within metal organic frameworks – to configure strong catalytic activity and robust protection. Upon target RNA hybridization, the transducer activates directly to liberate catalytic complexes, in a target-recyclable manner; when partitioned within a microfluidic device, these complexes can individually catalyze strong chemifluorescence reactions for digital RNA quantification. The EZ-READ platform thus enables programmable and reliable RNA detection, across different-sized RNA subtypes (miRNA and mRNA), directly in sample lysates. When clinically evaluated, the EZ-READ platform established composite signatures for accurate blood-based GBM diagnosis and subtyping.

P-745 Follicular fluid exposure may activate gene expression in mature human sperm before fertilization

Abstract Study question Does follicular fluid (FF) exposure shape human sperm RNA content and are these changes acquired through genomic or nongenomic manner? Summary answer FF exposure altered sperm RNA content potentially via activation of sperm RNA transcription machinery and caused selective intake of FF RNA transcripts in sperm. What is known already Mature sperm have been widely recognized as transcriptionally inert. Therefore, sperm RNAs have been regarded as the sole remnants of the gene expression process that occurs during spermatogenesis. Supporting this view, most sperm histones are replaced by protamines in developing spermatids to enable tighter packaging of the genetic material, which should prevent mature sperm cells to express their genes. However, in humans, approximately 15% of the sperm DNA remains associated with histones, raising the possibility that these genome regions could be available for de novo RNA transcription under an appropriate stimulus. Study design, size, duration Fresh, mature sperm were divided into three aliquots. One aliquot was used as an untreated control sample, the second and third aliquots were treated with either PureSperm® Wash (SW) solution or an FF pool of five females for three hours. RNA sequencing (RNA-seq) data was collected from FF and sperm samples. Results were complemented with Chromatin Immunoprecipitation Sequencing (ChIP-seq) data from human mature sperm (GEO: GSE15690 and GSE49624) and nascent RNA capture followed by PCR. Participants/materials, setting, methods All male and female participants were of reproductive age, non-smoking, healthy individuals who reported no history of infertility. Differentially expressed genes (DEG) were identified based on RNA-seq data (adjusted p-value < 0.05, logFC < |0.75|). The histone status of the genomic loci of DEGs was evaluated using ChIP-seq data. DEGs were also subjected to Gene Ontology analysis. Activated RNA transcription was confirmed by the Click-iT™ Nascent RNA Capture kit and PCR. Main results and the role of chance RNA-seq showed 193 DEGs between untreated and FF-treated samples of which 30 genes were upregulated and 163 were downregulated. Furthermore, 18 DEGs were detected between SW- and FF-treated sperm of which 17 genes were upregulated and one downregulated. Finally, 137 DEGs (all downregulated) were found between untreated and SW-treated samples. Among upregulated genes, chemokine-signaling, ion transport, and steroidogenesis-related biological functions were enriched whereas RNA processing, translation, and protein targeting-related biological processes were enriched among downregulated genes (untreated vs. FF-treated). ChIP-seq data revealed that 93% of upregulated DEGs are located within histone-bound chromatin and most of them bear both active and inactive histone markers. Finally, we confirmed that the upregulation of at least two genes (CXCL3 and CXCL8) resulted from active RNA transcription by sperm, whereas the upregulation of IGF2 was due to the selective internalization of mRNA from FF. Additionally, we identified 1396 protein-coding mRNAs present in human FF that were associated with gene expression-related biological processes such as the regulation of transcription, processing of RNA subtypes, and translation. Limitations, reasons for caution Due to the limited sample size, the generalizability of our results should be validated in the future. Furthermore, molecular mechanisms behind observed nascent RNA production needs to be clarified in more detail, including potential FF-mediated remodeling of sperm histone markers involved in the activation of gene expression. Wider implications of the findings Our findings demonstrate that FF exposure changes sperm RNA content, which seems to be partly caused by the active transcription of mature sperm, which has been widely believed to be non-existent. Therefore, our results can have many important implications for a deeper understanding of fertilization, early embryogenesis, and infertility. Trial registration number Not applicaple

Seminal extracellular vesicle sncRNA sequencing reveals altered miRNA/isomiR profiles as sperm retrieval biomarkers for azoospermia.

Non-invasive molecular biomarkers for classifying azoospermia by origin into either obstructive or non-obstructive/secretory azoospermia, as well as for inferring the spermatogenic reserve of the testis of non-obstructive/secretory azoospermia patients, are of great interest for testicular sperm retrieval outcome prediction for assisted reproduction. Prior analyses of semen small non-coding RNA expression in azoospermia have focused on microRNAs, but there has been a lack of attention on other regulatory small RNA species. In this regard, studying more in-depth expression changes of small non-coding RNA subtypes in small extracellular vesicles from semen of azoospermic individuals could be useful to select additional non-invasive biomarkers with diagnostic/prognostic purposes. A high-throughput small RNA profiling analysis to determine the expression pattern of seminal small extracellular vesicle microRNAs (analyzed at the isomiR level), PIWI-interacting RNAs, and transfer RNA-derived small RNAs in normozoospermic (n = 4) and azoospermic (obstructive azoospermia because of pathological occurring obstruction in the genital tract, n = 4; secretory azoospermic individuals with positive testicular sperm extraction value, n = 5; secretory azoospermic individuals with negative testicular sperm extraction value, n = 4) individuals was carried out. Reverse transcriptase-quantitative real-time polymerase chain reaction validation analysis of selected microRNAs was additionally performed in a larger number of individuals. Clinically relevant quantitative changes in the small non-coding RNA levels contained in semen small extracellular vesicles can be used as biomarkers for the origin of azoospermia and for predicting the presence of residual spermatogenesis. In this regard, canonical isoform microRNAs (n = 185) but also other isomiR variants (n = 238) stand out in terms of numbers and fold-change differences in expression, underlining the need to consider isomiRs when investigating microRNA-based regulation. Conversely, although transfer RNA-derived small RNAs are shown in our study to represent a high proportion of small non-coding RNA sequences in seminal small extracellular vesicle samples, they are not able to discriminate the origin of azoospermia. PIWI-interacting RNA cluster profiles and individual PIWI-interacting RNAs with significant differential expression were also not able to discriminate. Our study demonstrated that expression values of individual and/or combined canonical isoform microRNAs (miR-10a-5p, miR-146a-5p, miR-31-5p, miR-181b-5p; area under the receiver operating characteristic curve >0.8) in small extracellular vesicles provide considerable clinical value in identifying samples with a high likelihood of sperm retrieval while discriminating azoospermia by origin. Although no individual microRNA showed sufficient discriminating power on its own to identify severe spermatogenic disorders with focal spermatogenesis, multivariate microRNA models in semen small extracellular vesicles have the potential to identify those individuals with residual spermatogenesis. Availability and adoption of such non-invasive molecular biomarkers would represent a great improvement in reproductive treatment decision protocols for azoospermia in clinical practice.

Apicobasal RNA asymmetries regulate cell fate in the early mouse embryo

The spatial sorting of RNA transcripts is fundamental for the refinement of gene expression to distinct subcellular regions. Although, in non-mammalian early embryogenesis, differential RNA localisation presages cell fate determination, in mammals it remains unclear. Here, we uncover apical-to-basal RNA asymmetries in outer blastomeres of 16-cell stage mouse preimplantation embryos. Basally directed RNA transport is facilitated in a microtubule- and lysosome-mediated manner. Yet, despite an increased accumulation of RNA transcripts in basal regions, higher translation activity occurs at the more dispersed apical RNA foci, demonstrated by spatial heterogeneities in RNA subtypes, RNA-organelle interactions and translation events. During the transition to the 32-cell stage, the biased inheritance of RNA transcripts, coupled with differential translation capacity, regulates cell fate allocation of trophectoderm and cells destined to form the pluripotent inner cell mass. Our study identifies a paradigm for the spatiotemporal regulation of post-transcriptional gene expression governing mammalian preimplantation embryogenesis and cell fate.

Abstract 2806: Patient-derived cell-based pharmacogenomic platform to uncover resistance mechanisms and novel therapeutics for advanced lung cancer

Abstract To uncover resistance mechanism and effective drugs for advanced lung cancer patients, we stablished a pharmacogenomic platform using patient-derived cells (PDC). Drug response screening and multi-omics datasets were obtained from lung cancer PDCs (n=102). We interrogated sensitive drugs from variant, gene expression, and clinical profiles. Machine learning approaches were applied to extract molecular target signatures and pathways associated with sensitive drugs. Next, acquired resistance pathways and drug candidates were investigated from patients to receive serial EGFR-TKI treatments. Our PDC model resembled with TCGA genomic profile. Three RNA subtypes were identified from gene expression: (1) MYC, (2) epithelial-to-mesenchymal (EMT), and (3) inflammatory activation. Drugs for mutations and subtypes were extracted. Especially, SCLC harboring both TP53 and RB1 mutations was abundant in RNA subtype 1 and showed sensitivity to cell cycle inhibitors. Among EGFR-TKI treatment groups, Osimertinib resistant group was enriched in EMT-like subtype 2 with vanishing EGFR T790M mutation and activating TGF-β pathway. The group showed sensitivity to YAP/TAZ inhibitor. Our PDC pharmacogenetic platform predict plausible drug candidates and markers for refractory lung cancer patients. We expect further studies for precision medicine by novel analysis and cohort expansion. Citation Format: Charny Park, Namhee Yu, Mihwa Hwang, Youngjoo Lee, Bo Ram Song, Eun Hye Kang, Hanna Sim, Beung-Chul Ahn, Kum Hui Hwang, Sehwa Hong, Sunshin Kim, Ji-Youn Han. Patient-derived cell-based pharmacogenomic platform to uncover resistance mechanisms and novel therapeutics for advanced lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2806.

Systematic characterization and biological functions of non-coding RNAs in glioblastoma.

Glioblastoma multiforme (GBM) is the most malignant and aggressive type of glioma. Non-coding RNAs (ncRNAs) are RNAs that do not encode proteins but widely exist in eukaryotic cells. The common characteristics of these RNAs are that they can all be transcribed from the genome without being translated into proteins, thus performing biological functions, particularly microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs. Studies have found that ncRNAs are associated with the occurrence and development of GBM, and there is a complex regulatory network among ncRNAs, which can regulate cell proliferation, migration, apoptosis and differentiation, thus provide a basis for the development of highly specific diagnostic tools and therapeutic strategies in the future. The present review aimed to comprehensively describe the biogenesis, general features and functions of regulatory ncRNAs in GBM, and to interpret the potential biological functions of these ncRNAs in GBM as well as their impact on clinical diagnosis, treatment and prognosis and discusses the potential mechanisms of these RNA subtypes leading to cancer in order to contribute to the better design of personalized GBM therapies in the future.

Cloning and Sequencing Eukaryotic Small RNAs.

Small RNAs are ubiquitous regulators of gene expression that participate in nearly all aspects of physiology in a wide range of organisms. There are many different classes of eukaryotic small RNAs that play regulatory roles at every level of gene expression, including transcription, RNA stability, and translation. While eukaryotic small RNAs display diverse functions across and within classes, they are generally grouped functionally based on the machinery required for their biogenesis, the effector proteins they associate with, and their molecular characteristics. The development of techniques to clone and sequence small RNAs has been critical for their identification, yet the ligation-dependent addition of RNA adapters and the use of reverse transcriptase to generate cDNA in traditional library preparation protocols can be unsuitable to detect certain small RNA subtypes. In particular, 3' or 5' chemical modifications that are characteristic of specific types of small RNAs can impede the ligation-dependent addition of RNA adapters, while internal RNA modifications can interfere with accurate reverse transcription. The inability to clone certain small RNA subtypes with traditional protocols results in an inaccurate assessment of small RNA abundance and diversity, where some RNAs appear over-represented and others are not detected. This overview aims to guide users on how to design small RNA cloning workflows in eukaryotes to more accurately capture specific small RNAs of interest. Hence, we discuss the molecular biology underlying the identification and quantitation of small RNAs, explore the limitations of commonly used protocols, and detail the alternative approaches that can be used to enrich specific small RNA classes. © 2022 Wiley Periodicals LLC.

Exosomes in the Pathogenesis, Progression, and Treatment of Osteoarthritis.

Osteoarthritis (OA) is a prevalent and debilitating age-related joint disease characterized by articular cartilage degeneration, synovial membrane inflammation, osteophyte formation, as well as subchondral bone sclerosis. OA drugs at present are mainly palliative and do not halt or reverse disease progression. Currently, no disease-modifying OA drugs (DMOADs) are available and total joint arthroplasty remains a last resort. Therefore, there is an urgent need for the development of efficacious treatments for OA management. Among all novel pharmaco-therapeutical options, exosome-based therapeutic strategies are highly promising. Exosome cargoes, which include proteins, lipids, cytokines, and various RNA subtypes, are potentially capable of regulating intercellular communications and gene expression in target cells and tissues involved in OA development. With extensive research in recent years, exosomes in OA studies are no longer limited to classic, mesenchymal stem cell (MSC)-derived vesicles. New origins, structures, and functions of exosomes are constantly being discovered and investigated. This review systematically summarizes the non-classic origins, biosynthesis, and extraction of exosomes, describes modification and delivery techniques, explores their role in OA pathogenesis and progression, and discusses their therapeutic potential and hurdles to overcome in OA treatment.

Abstract P3-10-03: Enrichment of pro-oncogenic immune miRNAs in exosomes by cyclin D1, promote cancer stem cell expansion

Abstract The cyclin D1 (CCND1) gene, encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the Rb protein. Cyclin D1 overexpression occurs in up to 50% of human breast cancers. Cyclin D1, governs the expression (1,3), processing (2), the secretion and the relative proportion of secreted non-coding RNA subtypes (miRNA, rRNA, tRNA, CDBox, scRNA, HAcaBox. scaRNA, piRNA) in human breast cancer and the secretion of piRNA (4). Through base-pairing to the complementary sequence in 3’ untranslated region (3’ UTR) of mRNA, miRNAs participate in diverse biological processes. A subset of miRNA regulate the ageing of innate and adaptive immune cells, which has been associated with breast cancer initiation, progression and therapy resistance. Immuno-miRs, ( miR-21 and miR-93), bind to the 3′-UTR region of mRNAs, and binding to- and or activate Toll-Like Receptor 8 (TLR8) in surrounding cells of the immune system. Exosomes which contain constituents of their cell of origin, including mRNA and miRNA are found in the tumor microenvironment and participate in therapy resistance, cancer metastasis and the metastatic niche. Herein, tet-inducible cyclin D1 shRNA knock down in MCF-7 breast cancer cells reduced MCF-7 cell proliferation and mammosphere formation. Exosomes isolated from wildtype and cyclin D1 knock-down MCF-7 cell culture media, showed that exosomes from cyclin D1 knock down cells decreased proliferation of both wildtype and cyclin D1 knock-down cells. Furthermore, wildtype exosomes partially reversed the cyclin D1 knock-down effects. Cyclin D1 knock-down exosomes decreased mammosphere formation of wildtype MCF-7 cells and wildtype exosomes increased mammosphere formation of cyclin D1 knock-down cells. miRNA profiles within the exosomes, showed that cyclin D1 regulated the abundance of miRNA within exosomes. >85% of the cyclin D1-induced exosome miRNA transcripts were the immune-mIR, miR21. hsa-mir-21 which has documented tumor promoting function, was the most enriched miRNAs in cyclin D1 knock-down exosomes. In contrast, exosome abundance of the transcripts for hsa-mir-16 and hsa-mir-92a, both of which were showed as tumor suppressor in breast cancer cells, were reduced by cyclin D1 shRNA. The cyclin D1-mediated miRNA exosome content may contribute to tumor initiation and progression through heterotypic expansion of cancer stem cells. 1.Yu, Z., etal ., A cyclin D1/microRNA 17/20 regulatory feedback loop in control of breast cancer cell proliferation. J Cell Biol. 2008 Aug 11; 182(3): p. 509-17. 2.Yu., Z,., et. al. Cyclin D1 induction of Dicer governs microRNA processing and expression in breast cancer. Nat Commun 2013;4:2812.doi: 10.1038/ncomms3812. 3.Yu Z, et al. Cyclin D1- Mediated MicroRNA Expression Signature in Breast Cancer Subtype and Outcome. Theranostics, 2018 Mar 11,8:8, 2251-2263. 4.Lü J, et al. Cyclin D1 promotes secretion of pro-oncogenic immuno-miRNAs and piRNAs. Clin Sci (Lond). 2020 Mar 27. pii: CS20191318. doi: 10.1042/CS20191318. [Epub ahead of print]PMID:32219337 Citation Format: Xuanmao Jiao, Chongwen Xu, Lifeng Tian, Zhao Zhang, Anthony W Ashton, Zhiping Li, Richard G Pestell. Enrichment of pro-oncogenic immune miRNAs in exosomes by cyclin D1, promote cancer stem cell expansion [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-10-03.

Prognostic ability of the Gustave Roussy Immune Score for patients with advanced pancreatic adenocarcinoma.

469 Background: The Gustave Roussy Immune Score (GRIm-S) considers a composite of neutrophil to lymphocyte ratio (> 6 = 1), albumin (< 35 = 1) and LDH (> ULN = 1) and has been established as a prognostic score and may in aid in the selection of patients for phase 1 trials of immune checkpoint inhibitors. Methods: We explored the prognostic impact of the GRIm-S (high > 1) in patients enrolled on the COMPASS trial and correlated the score with genomic and clinical characteristics. Patients in this trial had biopsies for whole genomic and RNA sequencing prior to standard chemotherapy regimens in the advanced setting. Results: 252 patients were included in the analyses with a median follow-up time of 28 months. 16% of patients had a high GRIm-S with significantly shorter median overall survival (OS) of 4.1 months versus 10.0 months in those with a low score (HR 2.18, 95% CI 1.4-3.4, p < 0.0001). In the GRIm-S-high cohort, early progression with non-evaluable disease and disease progression were more common than in the GRIm-S low cohort (56% vs 31%, p = 0.003). In a multivariable analysis, a high GRIm-S was poorly prognostic (HR 1.6 95% CI 1.3-1.9, p < 0.001), whereas the classical RNA subtype (vs. basal-like) (HR 0.41, 95% CI 0.3-0.6, p < 0.001) and a high HRDetect score (HR 0.47 95% CI 0.3-0.7, p < 0.001) associated with superior OS. The GRIm-S did not correlate with RNA subtypes or with specific KRAS mutations. There were no differences in structural variant load or tumour mutational burden between groups. However those with a high GRIm-S did have a higher total target lesion diameter at baseline (p < 0.001). Conclusions: The GRIm-S identifies a subset of patients who have aggressive pancreas cancer and short life expectancy. This information may help clinicians in treatment decision making and selection for clinical trials.

Abstract PO-077: Establishment of a novel living biobank of patient-derived pancreatic cancer organoids with genomic and drug response characterization

Abstract Advanced pancreatic cancer has a dismal prognosis and current treatment options (FOLFIRINOX, Gemcitabine/nab-paclitaxel [GnP]) are associated with toxicity. Although some patients achieve partial responses, most progress rapidly and become chemorefractory. While RNA subtypes, genomic alterations, and protein biomarkers have prognostic value, predictive biomarkers to guide therapy are needed. Patient-derived organoids (PDOs) are an increasingly popular model for predicting patient responses to standard-of-care therapy and investigating personalized therapy options. We present a novel biobank of 42 PDOs and drug profiling data with 5 standard of care agents and 3 kinase inhibitors. Tissue was processed from n=103 biopsies from 97 patients with a confirmed pathologic diagnosis of advanced (Stage III-IV) pancreatic ductal adenocarcinoma who presented to a single Canadian tertiary care centre between 2017-2020. Matched WGS was available in all cases. Our PDO generation success was 42/103 (41%). We observed a trend towards decreased establishment in tumors that were KRAS WT, TP53 WT, or had higher HRDetect scores. Conversely, polyploidy, SMAD4 WT, and major imbalances in mutant KRAS were associated with successful PDO establishment. These associations were not statistically significant after multiple comparisons correction, but suggest selection for success with more aggressive tumors. Drug profiling was performed on all 42 PDOs with the individual agents of FOLFIRINOX (5-FU, irinotecan, oxaliplatin), GnP (gemcitabine, paclitaxel), and three targeted agents (afatinib, trametinib, and talazoparib). Combination testing was also performed for gemcitabine + paclitaxel. Drug responses were measured through both viability and growth rate (GRMetrics). We found that GRMetrics minimized effects from different PDO growth rates. Matched clinical data were available for 23 patients who received FOLFIRINOX, 11 patients who received GnP, and one patient who received gemcitabine monotherapy. Similar to previous studies, we found that in vitro PDO responses to 5-FU, irinotecan, and GnP were correlated with patient responses based on RECIST criteria. Interestingly, and similar to previous reports in colorectal cancer PDOs, we found that oxaliplatin responses were not predictive of RECIST response. As expected, PDOs were resistant to afatinib (EGFRi), which reflects negative clinical trials, and may also be masked by use of EGF in growth media. A range of responses to trametinib (MEKi) were seen but were not correlated with KRAS allelic dosage. A similar range of response was seen to talazoparib (PARPi), but did not correlate with oxaliplatin response or HRDetect scores. In summary, we have established a novel biobank of PDOs from advanced pancreatic cancer patients. Notably, PDOs were less likely to establish from tumors that were KRAS WT or HR-deficient, even though these patients are likely to benefit from targeted approaches. Further investigation is required to develop PDO use in clinical drug prediction and drug discovery. Citation Format: Irene Y. Xie, Laura Tamblyn, Karen Ng, Eugenia Flores-Figueroa, Julie M. Wilson, Gun Ho Jang, Amy X. Zhang, Stephanie Ramotar, Anna Dodd, Nikolina Radulovich, Jennifer J. Knox, Grainne M. O'Kane, Steven Gallinger, Faiyaz Notta. Establishment of a novel living biobank of patient-derived pancreatic cancer organoids with genomic and drug response characterization [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-077.

Dynamic phenotypic heterogeneity and the evolution of multiple RNA subtypes in hepatocellular carcinoma: the PLANET study.

Intra-tumor heterogeneity (ITH) is a key challenge in cancer treatment, but previous studies have focused mainly on the genomic alterations without exploring phenotypic (transcriptomic and immune) heterogeneity. Using one of the largest prospective surgical cohorts for hepatocellular carcinoma (HCC) with multi-region sampling, we sequenced whole genomes and paired transcriptomes from 67 HCC patients (331 samples). We found that while genomic ITH was rather constant across stages, phenotypic ITH had a very different trajectory and quickly diversified in stage II patients. Most strikingly, 30% of patients were found to contain more than one transcriptomic subtype within a single tumor. Such phenotypic ITH was found to be much more informative in predicting patient survival than genomic ITH and explains the poor efficacy of single-target systemic therapies in HCC. Taken together, we not only revealed an unprecedentedly dynamic landscape of phenotypic heterogeneity in HCC, but also highlighted the importance of studying phenotypic evolution across cancer types.

Abstract 2356: Cyclin D1 mediated exosomes are enriched for pro-oncogenic miRNAs and promote cancer stem cell expansion

Abstract miRNAs are a class of endogenous noncoding small RNAs that participate in diverse biological processes through base-pairing to the complementary sequence in 3' untranslated region (3' UTR) of mRNA Specific miRNA have been associated with the initiation, progression and therapy resistance and regulate immunological processes including the activation and ageing of innate and adaptive immune cells. Immuno-miRs, including. miR-21 and miR-93, regulate immune function in the tumor microenvironment by binding to the 3′-UTR region of target mRNAs, or by binding to- and activating Toll-Like Receptor 8 (TLR8) in surrounding cells of the immune system. The cyclin D1 (CCND1) gene, which encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the Rb protein, is overexpressed in up to 50% of human breast cancers. Cyclin D1, governs the expression, processing, the secretion and the relative proportion of secreted non-coding RNA subtypes (miRNA, rRNA, tRNA, CDBox, scRNA, HAcaBox. scaRNA, piRNA) in human breast cancer and the secretion of piRNA. Exosomes, which consist of a lipid bilayer membrane surrounding a small cytosol, contain constituents of their cell of origin, including mRNA and miRNA. Exosomes in the tumor microenvironment, participate in therapy resistance, cancer metastasis and the metastatic niche. Herein, knock down of cyclin D1 in MCF-7 cells with tet-inducible cyclin D1 shRNA decreased MCF-7 cell proliferation and mammosphere formation. Exosomes isolated from wild-type and cyclin D1 knock-down MCF-7 cell culture media, showed that exosomes from cyclin D1 knock down cells dramatically decreased proliferation of both wild-type and cyclin D1 knock-down cells, whereas the wild-type exosomes partially reversed the cyclin D1 knock down effects. Cyclin D1 knock-down exosomes decreased mammosphere formation of wild-type MCF-7 cells and wild-type exosomes increased mammosphere formation of cyclin D1 knock-down cells. miRNA profiles within the exosomes, showed that cyclin D1 regulated the abundance of miRNA within exosomes. The pattern of miRNA within exosomes regulated by cyclin D1 were enriched for miRNA governing mesenchymal epithelial transition and the induction of stem cell function. The most enriched miRNA in cyclin D1 wild-type exosomes was hsa-mir-21 which was recognized tumor promoting function, whereas the most enriched miRNAs in cyclin D1 knock-down exosomes were has-mir-16 and has-mir-92a, both of which were shown as tumor suppressor in breast cancer cells. The immune-miR miR-21 represented >85% of the cyclin D1-induced exosome miRNA transcripts, The cyclin D1-mediated miRNA exosome content may contribute to tumor initiation and progression through heterotypic expansion of cancer stem cells. Citation Format: Xuanmao Jiao, Chongwen Xu, Lifeng Tian, Zhao Zhang, Anthony W. Ashton, Zhiping Li, Richard G. Pestell. Cyclin D1 mediated exosomes are enriched for pro-oncogenic miRNAs and promote cancer stem cell expansion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2356.

Identification of pancreatic adenocarcinoma molecular subtypes on histology slides using deep learning models.

4141 Background: Pancreatic adenocarcinoma (PAC) is predicted to be the second cause of death by cancer in 2030 and its prognosis has seen little improvement in the last decades. PAC is a very heterogeneous tumor with preeminent stroma and multiple histological aspects. Omic studies confirmed its molecular heterogeneity, possibly one of the main factors explaining the failure of most clinical trials. Two and three transcriptomic subtypes of tumor cells and stroma respectively, were described with major prognostic and predictive implications. The tumor subtypes, Basal-like and Classical, have been shown by several groups to be predictive of the response to first line chemotherapy. As of today, these subtypes can only be defined by RNA profiling which is limited by the quantity and quality of the samples (formalin fixation and low cellularity) as well as by the analytical delay that may restrict its application in routine care. In addition, tumors may harbor a mixture of several subtypes limiting their interpretation using bulk transcriptomic approaches and thereby their clinical use. Here, we propose a multistep approach using deep learning models to predict tumor components and their molecular subtypes on routine histological preparations. Methods: 728 whole-slide digitized histological slides corresponding to 350 consecutive resected PAC from four centers with clinical and transcriptomic data were assembled and used as a discovery set. PAC from TCGA (n = 134) was used as a validation set. Tumor regions from slides of the discovery set were annotated to train a multistep deep learning model that first recognizes tumor tissue and then predicts tumor and stroma cells molecular subtypes assessed by the published PurIST algorithm. Results: The tumor detection model was very efficient (AUC = 0.98 in the TCGA validation cohort). In the discovery set, the Basal-like/Classical classification performance of the model by cross validation was 0.79 (AUC) and reached 0.86 when restricted to samples with a high-confidence RNA-defined molecular subtype.Subtypes defined by the model were independently associated with overall survival in multivariate analysis (HR = 2.56 [1.87 - 3.49], pval < 0.001), and association was higher relatively to PurIST RNA subtypes (HR = 1.60 [1.17 - 2.19] pval < 0.001). In the validation cohort, the model had an overall AUC of 0.82, and 0.89 in the subset of “subtype-pure” tumors. In addition to demonstrating the value of histology-based deep learning models for tumor subtyping in PAC, these results also show the limit of molecular-based subtyping in highly heterogeneous samples. Conclusions: This study provides the first PAC subtyping tool usable worldwide in clinical practice, finally opening the possibility of patient molecular stratification in routine care and clinical trials.

The non-coding epitranscriptome in cancer.

Post-synthesis modification of biomolecules is an efficient way of regulating and optimizing their functions. The human epitranscriptome includes a variety of more than 100 modifications known to exist in all RNA subtypes. Modifications of non-coding RNAs are particularly interesting since they can directly affect their structure, stability, interaction and function. Indeed, non-coding RNAs such as tRNA and rRNA are the most modified RNA species in eukaryotic cells. In the last 20 years, new functions of non-coding RNAs have been discovered and their involvement in human disease, including cancer, became clear. In this review, we will present the evidence connecting modifications of different non-coding RNA subtypes and their role in cancer.

Prevalence of hepatitis C virus genotypes in HIV positive patients referring to the consultation center for behavioral diseases, Sanandaj, Iran

Background and Objectives:Co-infection of hepatitis C virus (HCV) with human immunodeficiency virus (HIV) is increasing due to similar transmission pathways. Chronic HCV infection is the most common complication among HIV-infected individuals. Information on the frequency of HCV infection on Iranian HIV-infected individuals is scarce. The aim of this study was the detection of HCV prevalence and genotypes among HIV-infected people in Sanandaj, Iran.Materials and Methods:In this cross-sectional study, whole blood samples were taken from 185 HIV positive individuals referring to Consultation Center for Behavioral Diseases, Sanandaj, Iran. The ELISA test was done on samples for anti-HCV antibodies. RNA was extracted from only anti-HCV antibody positive samples. An RT-PCR test was conducted to detect HCV RNA. Genotypes of HCV were detected by melting curve analysis by specific primers and probes. Test results and demographic information were analyzed by SPSS software.Results:The mean age of individuals was 39.3 ± 9.4 years. Out of 185 individuals 99 (53.5%) were positive for anti-HCV antibodies. Out of 99 antibody positive individuals, 44 had HCV RNA. Among 44 RNA positive individuals, genotypes and subtypes of HCV were as 26 (59.1%) 1a, 17 (38.6%) 3a and one (2.2%) 4. There was a significant association between anti-HCV antibody and demographic variables including, age, gender, occupation, and CD4+ T-cell count (p = 0.0001).Conclusion:The present study reveals that HIV/HCV co-infection is high in the study population. It is recommended similar studies should be done in other HIV infected populations for management of HIV/HCV co-infection.

RNAcentral 2021: secondary structure integration, improved sequence search and new member databases.

RNAcentral is a comprehensive database of non-coding RNA (ncRNA) sequences that provides a single access point to 44 RNA resources and >18 million ncRNA sequences from a wide range of organisms and RNA types. RNAcentral now also includes secondary (2D) structure information for >13 million sequences, making RNAcentral the world’s largest RNA 2D structure database. The 2D diagrams are displayed using R2DT, a new 2D structure visualization method that uses consistent, reproducible and recognizable layouts for related RNAs. The sequence similarity search has been updated with a faster interface featuring facets for filtering search results by RNA type, organism, source database or any keyword. This sequence search tool is available as a reusable web component, and has been integrated into several RNAcentral member databases, including Rfam, miRBase and snoDB. To allow for a more fine-grained assignment of RNA types and subtypes, all RNAcentral sequences have been annotated with Sequence Ontology terms. The RNAcentral database continues to grow and provide a central data resource for the RNA community. RNAcentral is freely available at https://rnacentral.org.

RNA in cancer.

While the processing of mRNA is essential for gene expression, recent findings have highlighted that RNA processing is systematically altered in cancer. Mutations in RNA splicing factor genes and the shortening of 3' untranslated regions are widely observed. Moreover, evidence is accumulating that other types of RNAs, including circular RNAs, can contribute to tumorigenesis. In this Review, we highlight how altered processing or activity of coding and non-coding RNAs contributes to cancer. We introduce the regulation of gene expression by coding and non-coding RNA and discuss both established roles (microRNAs and long non-coding RNAs) and emerging roles (selective mRNA processing and circular RNAs) for RNAs, highlighting the potential mechanisms by which these RNA subtypes contribute to cancer. The widespread alteration of coding and non-coding RNA demonstrates that altered RNA biogenesis contributes to multiple hallmarks of cancer.

Cyclin D1 promotes secretion of pro-oncogenic immuno-miRNAs and piRNAs.

The molecular mechanisms governing the secretion of the non-coding genome are poorly understood. We show herein that cyclin D1, the regulatory subunit of the cyclin-dependent kinase that drives cell-cycle progression, governs the secretion and relative proportion of secreted non-coding RNA subtypes (miRNA, rRNA, tRNA, CDBox, scRNA, HAcaBox. scaRNA, piRNA) in human breast cancer. Cyclin D1 induced the secretion of miRNA governing the tumor immune response and oncogenic miRNAs. miR-21 and miR-93, which bind Toll-Like Receptor 8 to trigger a pro-metastatic inflammatory response, represented >85% of the cyclin D1-induced secreted miRNA transcripts. Furthermore, cyclin D1 regulated secretion of the P-element Induced WImpy testis (PIWI)-interacting RNAs (piRNAs) including piR-016658 and piR-016975 that governed stem cell expansion, and increased the abundance of the PIWI member of the Argonaute family, piwil2 in ERα positive breast cancer. The cyclin D1-mediated secretion of pro-tumorigenic immuno-miRs and piRNAs may contribute to tumor initiation and progression.