eRapport

MicroRNA i Colorectal Cancer

Prosjekt
Prosjektnummer
2014041
Ansvarlig person
Kjersti Flatmark
Institusjon
Oslo universitetssykehus HF
Prosjektkategori
Postdoktorstipend
Helsekategori
Cancer
Forskningsaktivitet
1. Underpinning, 4. Detection and Diagnosis
Rapporter
2019 - sluttrapport
MicroRNAs (miRNA) are short, endogenous RNA molecules that regulate gene expression in the cell. Dysregulation of miRNAs is a key player in cancer progression, with miRNAs being involved in all hallmarks of cancer. Unlike other RNA molecules, miRNAs are highly stable and combined with robust and widely available methods for quantification, miRNAs hold excellent biomarker potential. However, after years of research, no miRNA biomarker is still in clinical use in colorectal cancer (CRC) management. There are many reasons for this lack of progress, however, one of them likely stems from the lack of a consensus methodology in the miRNA research community. Two main achievements have been accomplished in this project Establishment of a novel miRNA database available to the scientific community An important issue was the lack of a commonly accepted complete repository of miRNA genes with consistent nomenclature. A large number of false positives, false negatives, and duplicates were registered in the most commonly used database, miRBase. As a part of this project, our group established a new curated database consisting of only bona fide miRNA, containing data from four species, MirGeneDB (Fromm et al, Annu Rev Genet. 2015;49:213-42), and the database was in 2019 expanded to comprise 45 species in MirGeneDB2.0 (Fromm et al Nucleic Acids Res. 2020 Jan 8;48(D1):D132-D141). Establishment of this database that is available to the entire scientific community represents important ground-breaking work in the miRNA field. Generation of a specific miRNA signature for metastatic CRC Classification of the metastatic CRC (mCRC) miRNA transcriptome to identify potential miRNA biomarkers of mCRC has proven challenging for many reasons. A critical issue in miRNA research is that the vast majority of data is based on data derived from bulk tissue. Although miRNAs have been widely reported to be tissue-type specific, it is important to keep in mind that the specificity is related to expression at a cellular level. Since tissues are made up of many different cell types, alterations in the cellular composition of tissues during disease, such as cancer, will very likely lead to changes in the expression of a cell-specific miRNA when comparing healthy and diseased tissues. For example, since CRC is derived from epithelial cells, the apparent “downregulation” of non-epithelial miRNA in CRC compared to normal tissues can most likely be explained by differences in cellular composition, not by changes in expression in the tumor cells. Another important point in miRNA research is that when handling data from next-generation sequencing (NGS), it is critical that the bioinformatics analysis of all datasets is performed in the same manner across datasets, using the same tools and parameters. To address these issues and identify a comprehensive miRNA signature for mCRC, our research group has analyzed our own NGS datasets with publicly available datasets. We performed NGS using samples from our biobanks of primary and metastatic CRC (metastases to liver, lungs and peritoneum). Using a reproducible bioinformatics pipeline and MirGeneDB2.0 as the Reference across all datasets, we combined our analytical approach with previously published information on the cellular profile of miRNAs. This approach enabled identification of a comprehensive profile of miRNA expression in samples from primary CRC and metastatic tissues compared to their respective normal counterparts. Distinct miRNA profiles could distinguish the individual metastatic locations. A key finding was that Mir-210, which is upregulated during hypoxic conditions, was upregulated in lung and liver metastasis, compared to primary CRC. A manuscript with these results will be submitted spring 2020. The establishment of the new databases do not have direct implications for the health services. However, creation and public availability of the novel database MirGeneDB with the extension to MirGeneDB2.0 represent important progress in the miRNA field. To illustrate this, the two MirGeneDB publications have already been cited a total of 262 times (https://scholar.google.no/citations?user=rIFbpm0AAAAJ&hl=en), with a number of leading researchers in the field having switched from using miRBase to MirGeneDB as a reference. Therefore, although the clinical translational value may not be apparent, the work has had major impact and will provide an important tool for future miRNA research. The mCRC miRNA signatures have identified miRNAs that are likely to be involved in the metastatic process and may represent potential future biomarkers. The reproducible bioinformatics pipeline that was used to develop the signatures will be made available to other researchers as part of the published manuscript and will provide a standard for such analyses in the future when new datasets are generated.

Nei

2018
MicroRNAs are involved in all hallmarks of cancer and are promising cancer biomarkers. In this study we developed novel tools and using these, identified distinct signatures specific to metastatic colorectal cancer that add to biological understanding of colorectal cancer and may serve as biomarkers of metastatic disease.In colorectal cancer (CRC), the third most prevalent cancer worldwide, a large proportion of patients will develop metastases, which is the dominating cause of CRC-related mortality. We previously investigated the microRNA (miRNA) transcriptome of primary CRC (pCRC) using deep sequencing, and interesting findings were made regarding expression levels and clinical relevance. In this project we have investigated the miRNA transcriptome of metastatic CRC (mCRC) and developed novel bioinformatics tools for this purpose. The miRNA field is methodologically very challenging. An important part of this work has therefore been to develop necessary, reliable tools to ensure the validity of the analyses. Postdoctoral fellow Bastian Fromm has been co-associated with our department's bioinformatics group headed by professor Eivind Hovig, and the miRNA bioinformatics pipeline MirMiner for accurate prediction and expression analyses of miRNAs was further developed and tested. Also, the second version of the MirGeneDB2.0 database, the new standard for miRNA annotations with more than 75 citations since its publication November 2015 was finalized (manuscript submitted). Through a range of collaborative activities within and outside OUS our team contributed to a number of scientific publications in the cancer and non-coding RNA field. Deep sequencing of miRNA transcriptomes in mCRC and pCRC was performed on samples from our extensive biobanks. The results were combined with all available datasets from previously published studies into a large meta-analysis of pCRC and mCRC. For the first time, we identified distinct patterns of miRNA expression associated with mCRC located in different organs (liver, lung, peritoneum), comparing with normal tissues from the corresponding organs. Our in-house delveloped tools were used, and employing a strict and transparent bioinformatics processing pipeline, considering tumor heterogeneity and the latest results in the cellular miRNA expression patterns, we were able to identify miRNA expression patterns associated with metastatic location. Interestingly, the identified signatures contained miRNAs associated with immune cell infiltration that may have important clinical implications. The results were presented by Fromm at the annual meeting of the American Association for Cancer Research 2017 and a manuscript is being finalized for publication. Fromm who held the position that was funded by the project has moved on to a position in another research group, and the work is being completed in collaboration with him by other group members. Work is ongoing to validate the signature that was identified, and top candidate signature miRNAs will be analyzed by qPCR in mCRC samples from liver, lung and peritoneal metastases (n>500). This work is necessary in order to ensure publication of the results in a high-impact journal, which justifies the delay in publishing results from the project.

Nei

2017
MicroRNAs are involved in all hallmarks of cancer and are promising cancer biomarkers. In this study we developed novel tools and using these, identified distinct signatures specific to metastatic colorectal cancer that add to biological understanding of colorectal cancer and may serve as biomarkers of metastatic disease.In colorectal cancer (CRC), the third most prevalent cancer worldwide, a large proportion of patients will develop metastases, which is the dominating cause of CRC-related mortality. We previously investigated the microRNA (miRNA) transcriptome of primary CRC (pCRC) using deep sequencing, and interesting findings were made regarding expression levels and clinical relevance. In this project we have investigated the miRNA transcriptome of metastatic CRC (mCRC) and developed novel bioinformatics tools for this purpose. The miRNA field is methodologically very challenging. An important part of this work has therefore been to develop necessary, reliable tools to ensure the validity of the analyses. Postdoctoral fellow Bastian Fromm has been co-associated with our department's bioinformatics group headed by professor Eivind Hovig, and the miRNA bioinformatics pipeline MirMiner for accurate prediction and expression analyses of miRNAs was further developed and tested. Also, the second version of the MirGeneDB2.0 database, the new standard for miRNA annotations with more than 75 citations since its publication November 2015 was finalized (manuscript submitted). Through a range of collaborative activities within and outside OUS our team contributed to a number of scientific publications in the cancer and non-coding RNA field. Deep sequencing of miRNA transcriptomes in mCRC and pCRC was performed on samples from our extensive biobanks. The results were combined with all available datasets from previously published studies into a large meta-analysis of pCRC and mCRC. For the first time, we identified distinct patterns of miRNA expression associated with mCRC located in different organs (liver, lung, peritoneum), comparing with normal tissues from the corresponding organs. Our in-house delveloped tools were used, and employing a strict and transparent bioinformatics processing pipeline, considering tumor heterogeneity and the latest results in the cellular miRNA expression patterns, we were able to identify miRNA expression patterns associated with metastatic location. Interestingly, the identified signatures contained miRNAs associated with immune cell infiltration that may have important clinical implications. The results were presented by Fromm at the annual meeting of the American Association for Cancer Research 2017 and a manuscript is being finalized for publication. Plans have been made for further validation of the signatures in serum samples through a collaboration agreement with the Janus serum bank for access to a significant number of miRNA sequencing datasets. Additionally, top candidate signature miRNAs will be analyzed by qPCR in mCRC samples from liver, lung and peritoneal metastases (n>500).

NEI

2016
Colorectal cancer (CRC) is one of the leading causes of cancer related deaths and the search for prognostic biomarkers that might improve diagnosis and treatment decisions is warranted. MicroRNAs (miRNAs), a large class of short gene regulators, have been proposed as biomarkers in CRC and might hold the key to understand CRC and cancer in general.In colorectal cancer (CRC), the third most prevalent cancer worldwide, the majority of patients will develop metastases which is the dominating cause of CRC-related deaths. In earlier work we investigated the miRNA transcriptome of primary CRC using deep sequencing, and interesting findings were made regarding expression levels and clinical relevance. In this project we investigate the presence of signature miRNAs in metastatic CRC and have developed novel bioinformatics pipelines (MirGeneDB.org, 2015) for miRNA analyses. Progress in the three subprojects through 2016: Subproject 1: Further development of miRNA bioinformatics pipelines MirMiner and MirAthon for the accurate prediction and expression analyses of miRNAs. Postdoctoral fellow Bastian Fromm was invited to contribute a bookchapter on miRNA prediction and expression analysis that was published with Springer International Publishing ("Field Guidelines for Genetic Experimental Designs in High-Throughput Sequencing"). Progress and development of the pipeline ("Taming the beast—Towards a systematic framework for the discovery, accurate annotation, curation and expression analysis of microRNAs and their isoforms in cancer") was reported at an international meeting (Conference: Regulatory & Non-coding RNAs, Cold Spring Harbor) where it received very positive feedback. A beta-version of the pipeline was used for a masters project that was completed in 2016, resulting in the thesis "MicroRNAs in Metastases" by Eirik Høye, MSc. Subproject 2/3: Generate smallRNA datasets from metastatic CRC We have performed deep sequencing of miRNA transcriptomes in CRC liver metastases and resequenced primary tumors of CRC (subproject 2) and we combined these datasets with results from a larger meta-analyses of published miRNA and mRNA datasets. For the first time, we have identified a distinct pattern of miRNAs, a miRNA signature, associated with liver metastases and target mRNA (gene-expression data). Results will be presented at the annual meeting of the American Association for Cancer Research 2017 and a manuscript is in preparation. For subproject 2/3, we have also sequenced and analyzed miRNA transcriptomes of CRC metastases to the lung and peritoneal surfaces and normal tissues from the corresponding organs and are inthe process of validating the findings. Briefly, we identified distinct miRNA expression patterns associated with metastatic location. Subproject 2/3: Validation of results in other/extended cohorts of cancer patients We have established a collaboration with the Norwegian Cancer Registry, specifically regarding samples from the Janus serum bank, where we wish to attempt to validate our metastasis specific signature miRNAs in serum samples from cancer patients. We also will use available and newly generated RNA samples from the Oslo CoMet-study for qPCR validation experiments.
2015
Colorectal cancer (CRC) is one of the leading causes of cancer related deaths and the search for prognostic biomarkers that might improve diagnosis and treatment decisions is warranted. MicroRNAs (miRNAs), a large class of short gene regulators, have been proposed as biomarkers in CRC and might hold the key to understand CRC and cancer in general.CRC is a heterogeneous disease characterized by the accumulation of genetic and epigenetic events which is often influenced by lifestyle. Today, treatment decisions are essentially based on the anatomical extent of disease at diagnosis, and the search for better biomarkers is warranted. In earlier work we investigated the microRNA transcriptome of primary CRC using deep sequencing, and interesting findings were made regarding expression levels and clinical relevance. Among the many available techniques, deep sequencing has emerged as the most inclusive approach for global miRNA analysis, advantages include the wide detectable expression range, the ability to analyze expression of known and novel miRNAs and their isoforms and the possibility to scale sequencing depth by demand. However, deep sequencing comes with a usually neglected bioinformatics challenge: massive amounts of data need to be analyzed and compared to the state-of the art in miRNA research/knowledge. With the large amount of data that comes from each deep sequencing study, it has become difficult determining what is and what is not a miRNA. Many authors have argued that miRBase, the online repository for miRNAs, is riddled with false positives, i.e., sequences that are not derived from bona fide (= trustworthy) miRNA genes. By reviewing the existing data deposited in miRBase, we have established a consistent set of criteria for miRNA annotation, and when applied to the 1881 human miRNA entries listed in miRBase, we found that less than a third of these entries can be supported as bona fide miRNA genes. Together with international collaborators we developed a uniform system for the annotation and nomenclature of human miRNAs and reconstructed human miRNA evolution in an unprecedented manner. We have constructed the MirGeneDB.org, a publicly available database of our curated set of human miRNAs that will be useful for many researchers in the field. The manuscript was published in December 2015 in the prestigious journal Annual Reviews of Genetics and represents the basis for the future work in this project. The work got attention in several blogs, news outlets and has been included in the elixir tools and data services registry (1-5). For subproject 1 we have established national and international collaborations that aim at the creation of a comprehensive bioinformatics workflow for the fully automatized high-throughput analysis of smallRNAseq datasets on an unprecedented level. We will to complete and publish the pipeline in 2016, and will initially use it on our previously published data, on resequenced samples (in higher depth) as well as on new samples (subproject 2). As we have worked with the creation of a workflow not restricted to CRC, we have recently also gained access to datasets from all major kind of cancers that we in parallel analyze as a control to CRC and plan to publish separately. For subproject 2/3, we will analyze microRNA transcriptomes of CRC derived metastases (liver, lung and peritoneal metastases) and normal tissues from the corresponding organs (liver and lung). Samples are being processed, and a set of liver metastasis/normal liver samples was recently submitted for sequencing, and data will be available for analysis spring 2016. A master student has been recruited and will work on this data when available.
2014
Colorectal cancer (CRC) is one of the leading causes of cancer related deaths and the search for prognostic biomarkers that might improve diagnosis and treatment decisions is warranted. MicroRNAs (miRNAs), a large class of short gene regulators, have been proposed as biomarkers in CRC and might hold the key to understand CRC and cancer in general.CRC is a heterogeneous disease characterized by the accumulation of genetic and epigenetic events which is often influenced by lifestyle. Today, treatment decisions are essentially based on the anatomical extent of disease at diagnosis, and the search for better biomarkers is warranted. In earlier work we investigated the microRNAome of primary CRC samples using deep sequencing, and interesting findings were made regarding expression levels and clinical relevance. Among the many available techniques, deep sequencing has indeed emerged as the most inclusive approach for global miRNA analysis. Advantages include the wide detectable expression range, the ability to analyze expression of known and novel miRNAs and their isoforms and the possibility to scale sequencing depth by demand. However, deep sequencing comes with a bioinformatics challenge, as massive amounts of data need to be analyzed and compared to the state-of-the-art in miRNA research. Also, with miRNAs having taken center stage in dissection of human biology and diseases such as cancer, the huge number of scientific publications on miRNAs (35000) and on miRNA and cancer alone (3500) make defining state-of-the-art a daunting task. Importantly, it became clear that although miRNAs are in principle a well defined RNA species, the only available reference for known miRNAs, miRBase, is riddled with false positives, i.e. sequences that are not derived from bona fide (= trustworthy) miRNA genes, including many human entries. In order to put the whole project on a more solid foundation we reviewed the existing data deposited in miRBase. Together with international collaborators we compiled a comprehensive review about the field where we developed a uniform system for the annotation and nomenclature of human miRNAs. When we applied those criteria to the 1881 human miRNA entries listed in miRBase, we found that less than a third of all entries can be supported as bona fide miRNA genes. We created the miRNA gene database MirGeneDB.org that holds all confirmed genes, including and updated annotation to the human genome, and will provide a valuable resource for the project and many other researches. The resulting manuscript will be submitted as per invite by a leading journal of the field on the 15th of February 2015. Since the start of the project in August 2014 we also began to screen all available cohorts of primary and metastatic CRC for their suitability and sampling progress for a comprehensive smallRNA sequencing study with focus on the detection on biomarkers and miRNA isoforms. A comparison of different possible extraction methods and sampling approaches for the different tissue types was initiated and a Master student was recruited to the project, who will start in summer. A reanalysis of previously sequenced primary tumors of CRC based on the newly compiled set of bona fide miRNAs was performed and compared to newly generated data from non-cancer samples (colorectum).
Vitenskapelige artikler
Umu SU, Langseth H, Bucher-Johannessen C, Fromm B, Keller A, Meese E, Lauritzen M, Leithaug M, Lyle R, Rounge TB

A comprehensive profile of circulating RNAs in human serum.

RNA Biol 2018 02 01;15(2):242-250. Epub 2017 des 8

PMID: 29219730

Halushka MK, Fromm B, Peterson KJ, McCall MN

Big Strides in Cellular MicroRNA Expression.

Trends Genet 2018 03;34(3):165-167. Epub 2018 jan 18

PMID: 29361313

Tarver JE, Taylor RS, Puttick MN, Lloyd GT, Pett W, Fromm B, Schirrmeister BE, Pisani D, Peterson KJ, Donoghue PCJ

Well-Annotated microRNAomes Do Not Evidence Pervasive miRNA Loss.

Genome Biol Evol 2018 06 01;10(6):1457-1470.

PMID: 29788279

Fromm B, Tosar JP, Lu Y, Halushka MK, Witwer KW

Human and Cow Have Identical miR-21-5p and miR-30a-5p Sequences, Which Are Likely Unsuited to Study Dietary Uptake from Cow Milk.

J Nutr 2018 Sep 01;148(9):1506-1507.

PMID: 30184228

Bhambri A, Dhaunta N, Patel SS, Hardikar M, Bhatt A, Srikakulam N, Shridhar S, Vellarikkal S, Pandey R, Jayarajan R, Verma A, Kumar V, Gautam P, Khanna Y, Khan JA, Fromm B, Peterson KJ, Scaria V, Sivasubbu S, Pillai B

Large scale changes in the transcriptome of Eisenia fetida during regeneration.

PLoS One 2018;13(9):e0204234. Epub 2018 sep 27

PMID: 30260966

Fromm B, Kang W, Rovira C, Cayota A, Witwer K, Friedländer MR, Tosar JP

Plant microRNAs in human sera are likely contaminants.

J Nutr Biochem 2018 Sep 05. Epub 2018 sep 5

PMID: 30266285

Bråte J, Neumann RS, Fromm B, Haraldsen AAB, Tarver JE, Suga H, Donoghue PCJ, Peterson KJ, Ruiz-Trillo I, Grini PE, Shalchian-Tabrizi K

Unicellular Origin of the Animal MicroRNA Machinery.

Curr Biol 2018 Oct 22;28(20):3288-3295.e5. Epub 2018 okt 11

PMID: 30318349

Rodríguez M, Bajo-Santos C, Hessvik NP, Lorenz S, Fromm B, Berge V, Sandvig K, Line A, Llorente A

Identification of non-invasive miRNAs biomarkers for prostate cancer by deep sequencing analysis of urinary exosomes.

Mol Cancer 2017 Oct 05;16(1):156. Epub 2017 okt 5

PMID: 28982366

Graudenzi A, Cava C, Bertoli G, Fromm B, Flatmark K, Mauri G, Castiglioni I

Pathway-based classification of breast cancer subtypes.

Front Biosci (Landmark Ed) 2017 06 01;22():1697-1712. Epub 2017 jun 1

PMID: 28410140

Simovski B, Vodák D, Gundersen S, Domanska D, Azab A, Holden L, Holden M, Grytten I, Rand K, Drabløs F, Johansen M, Mora A, Lund-Andersen C, Fromm B, Eskeland R, Gabrielsen OS, Ferkingstad E, Nakken S, Bengtsen M, Nederbragt AJ, Thorarensen HS, Akse JA, Glad I, Hovig E, Sandve GK

GSuite HyperBrowser: integrative analysis of dataset collections across the genome and epigenome.

Gigascience 2017 Jul 01;6(7):1-12.

PMID: 28459977

Fromm B, Ovchinnikov V, Høye E, Bernal D, Hackenberg M, Marcilla A

On the presence and immunoregulatory functions of extracellular microRNAs in the trematode Fasciola hepatica.

Parasite Immunol 2017 Feb;39(2).

PMID: 27809346

Ovchinnikov VY, Mordvinov VA, Fromm B

Extreme conservation of miRNA complements in opisthorchiids.

Parasitol Int 2017 Dec;66(6):773-776. Epub 2017 sep 12

PMID: 28916307

Haakensen VD, Nygaard V, Greger L, Aure MR, Fromm B, Bukholm IR, Lüders T, Chin SF, Git A, Caldas C, Kristensen VN, Brazma A, Børresen-Dale AL, Hovig E, Helland Å

Subtype-specific micro-RNA expression signatures in breast cancer progression.

Int J Cancer 2016 Sep 01;139(5):1117-28. Epub 2016 mai 9

PMID: 27082076

Flatmark K, Høye E, Fromm B

microRNAs as cancer biomarkers.

Scand J Clin Lab Invest Suppl 2016;245():S80-3. Epub 2016 aug 9

PMID: 27542003

Ramalho-Carvalho J, Fromm B, Henrique R, Jerónimo C

Deciphering the function of non-coding RNAs in prostate cancer.

Cancer Metastasis Rev 2016 06;35(2):235-62.

PMID: 27221068

Fromm B, Billipp T, Peck LE, Johansen M, Tarver JE, King BL, Newcomb JM, Sempere LF, Flatmark K, Hovig E, Peterson KJ

A Uniform System for the Annotation of Vertebrate microRNA Genes and the Evolution of the Human microRNAome.

Annu Rev Genet 2015 Nov 23;49():213-42. Epub 2015 okt 14

PMID: 26473382

Deltagere
  • Svein Dueland Prosjektdeltaker
  • Kjersti Flatmark Hovedveileder
  • Leonardo Zepeda Meza Prosjektdeltaker
  • Susanne Lorenz Prosjektdeltaker
  • Bjørn Edwin Prosjektdeltaker
  • Annette Torgunrud Kristensen Prosjektdeltaker
  • Lund-Andersen Christin Prosjektdeltaker
  • Anne Hansen Ree Prosjektdeltaker
  • Øystein Fodstad Prosjektdeltaker
  • Diana Domanska Prosjektdeltaker
  • Johannes Eivind Hovig Medveileder
  • Bastian Fromm Postdoktorstipendiat (finansiert av denne bevilgning)
  • Eirik Høye Prosjektdeltaker

eRapport er utarbeidet av Sølvi Lerfald og Reidar Thorstensen, Regionalt kompetansesenter for klinisk forskning, Helse Vest RHF, og videreutvikles av de fire RHF-ene i fellesskap, med støtte fra Helse Vest IKT

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