Mechanistic and therapeutic implications of microRNA deregulation for drug resistance in bile duct cancer

Informations projet

MiRCHOL

N° de convention de subvention: 746068

DOI

10.3030/746068

Projet clôturé

Date de signature de la CE 15 Mars 2017

Date de début 1 Decembre 2017

Date de fin 23 Juillet 2020

Financé au titre de

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Coût total

€ 200 194,80

Contribution de l’UE

€ 200 194,80

Coordonné par

KOBENHAVNS UNIVERSITET
Denmark

Periodic Reporting for period 1 - MiRCHOL (Mechanistic and therapeutic implications of microRNA deregulation for drug resistance in bile duct cancer)

Période du rapport: 2017-12-01 au 2019-11-30

Résumé du contexte et des objectifs généraux du projet

Cholangiocarcinoma (CCA) is a dismal type of liver cancer, which incidence has more than doubled the last 20 years and mortality rate in Europe alone increased 37%, reflecting the limited treatment options. In >70% of patients CCA is unresectable due to advanced stage of disease at diagnosis, resulting in a 5% 5-year survival rate. CCA is characterized by molecular and clinical heterogeneity, complicating therapy and assessment of drug efficacy. Tumor heterogeneity is emphasized in limited response of therapy (drug resistance) and besides genomic instability a likely consequence of deregulated pathways under control of microRNAs (miRs). Each miR can regulate multiple genes/pathways, directly linking them to tumor heterogeneity. Deregulation of miRs can cause/control chemoresistance in other cancers, but in CCA their role is yet unclear. Thus, detailed characterization of miRs in CCA, focusing on their impact in drug resistance is urgently needed.

I had 3 specific aims of this proposal:

1) characterize the deregulated expression pattern of miRs involved in CCA pathogenesis and integrate this data with transcriptomes obtained from matched patients;
2) determine the role of deregulated miRs in control of chemoresistance, and
3) modulate by genome-editing key deregulated miR(s) targeting chemoresistant genes using the novel `CRISPR-Mir’ method.

Conclusions of the study:

I have generated the largest miRNA-transcriptome CCA dataset and identified miRNA-27a-3p/FoxO1 interaction as key therapeutic target in CCA for the first time.

Achievement of these aims has yield key new insights into the role of miRs in orchestrating CCA progression.

My findings will have an impact on the society and in particular in patient outcome.

Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent

Overall assessment: The project has achieved its main objectives and milestones.

Objective 1:
Aimed to characterize the deregulated expression pattern of miRs involved in the CCA pathogenesis and integrate this data with transcriptomic landscapes obtained from matched patients. Specific steps taken to achieve this objective included:
• Construction, analysis and interpretation of small RNAseq (miR) data set across a cohort of fresh frozen tissue samples, which included 22 normal/disease-free (NL), 63 surrounding normal (SL) and 120 resected CCA comprising 88 intrahepatic (iCCA), 13 perihilar (pCCA) and 19 distal (dCCA) samples.
• Integration of miRNA and gene expression data sets from matched patients using in parallel 2 primary CCA cell models, primary normal human cholangiocytes (NHC, control) and a panel of 20 well-characterized and established human CCA cell lines to account for molecular heterogeneity implicit in CCA.

Objective 2:
Sought to determine the role of deregulated miRNAs in control of chemoresistance of CCA. This objective was pursued through the following steps:
• Identification of candidate miRNAs in NHC (specifically NHC-3), evaluating the potential proliferation ability of 2,754 microRNA mimics (miRBase version 21 library).
• Validation of significant miRNAs found in the first screen in another 2 NHCs (C-324 and NHC-2).
• Examination of key miRNAs regulating the CCA transcriptome and causing drug resistance. Due to the important results found in tasks 2.1 and 2.2 the drug resistance assays will be performed outside the duration of the fellowship.

Objective 3:
Was designed to modulate by genome-editing key deregulated miRNAs targeting chemoresistant genes using the novel `CRISPR-Mir’ method. Thus, this objective includes:
• Successful CRISPR Knock-Out (KO) of miR-27a-3p, as validated in different in vitro assays.
• In vivo assessment of therapeutic efficacy of CRISPR-miR_27a-3p inflection. However, due to the necessary adjustments of WP2, this objective has not yet been fully finished but will performed outside the duration of the fellowship.

Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent)

The main research completed through the MiRCHOL project is highly inter-disciplinary. The project included in vitro experiments, kinetics, genomics and bioinformatics, and state-of-the-art genome editing tools (featuring high-throughput mimic screening and an advanced CRISPR-miRNA approach). Moreover, it is extremely novel as I have generated the largest miRNA-transcriptome CCA dataset and identified miRNA-27a-3p/FoxO1 interaction as key therapeutic target in CCA for the first time. Overall, the accomplishment of this project holds significant translational potential.

Completion of this fellowship has significantly advanced my research career in numerous ways including dramatically increasing my scientific and professional skillsets, enhancing my knowledge of translational oncology, expanding my professional network, and affording me opportunities to perform cutting-edge experiments, which ultimately have led to high impact publications and awards at international conferences.

I have engaged in numerous successful external collaborations during the course of this fellowship. These include international collaborations (Dr. Chiara Braconi, Edimburg; Prof. Jens Marquardt, Germany; Dr Jose Juan García Marín, Spain; Dr. Jung Chin Chang, The Netherlands). Accordingly, I have extensively expanded my professional network through completion of this fellowship in the host laboratory.

In reflection of these career advances I have made during my MSCA fellowship, I have been awarded with several recognized Spanish Grants, which open a window to take the next step on my career. These include Ikerbasque Research Fellow, Asociación Española Contra el Cáncer (AECC) and Juan de la Cierva (scored 98 out of 100 points, from the Spanish Ministry of Health and Research).

The work executed during the course of this fellowship holds significant potential to advance clinical management of CCA patients. The wider socio-economic implications of the MiRCHOL project may be considered once we obtain the last results from the in vivo study. However, socially I expect that our data will support the therapeutic efficacy of regulating/controlling a single miRNA in CCA, making miRNA therapy a very promising clinical treatment option for CCA patients, which currently lack of major options.

Summary of MIRCHOL project