EP4436584A1 - Treatment of rhabdomyosarcoma - Google Patents

Treatment of rhabdomyosarcoma

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Publication number
EP4436584A1
EP4436584A1 EP22823350.8A EP22823350A EP4436584A1 EP 4436584 A1 EP4436584 A1 EP 4436584A1 EP 22823350 A EP22823350 A EP 22823350A EP 4436584 A1 EP4436584 A1 EP 4436584A1
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Prior art keywords
mir
rms
cells
tumour
treatment
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German (de)
French (fr)
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Enrico Pozzo
Maurilio SAMPAOLESI
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Katholieke Universiteit Leuven
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Katholieke Universiteit Leuven
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/31—Combination therapy

Definitions

  • the invention relates to treatment of cancers with miRNAs.
  • Rhabdomyosarcoma occurs in 50% of paediatric soft tissue sarcomas and accounts for 3-4% of all paediatric cancers. The incidence is high below the age of 20, after which the incidence decreases to 1% of adult malignancies. In most cases, paediatric RMS tumours occur in the head and neck region.
  • a combination of oncosuppressor microRNAs is identified and their effects on the tumorigenicity and the metabolism of pyruvate in FN-RMS were characterized.
  • the present invention investigates the effect of mitochondrial pyruvate carrier (MPC) inhibition in human and murine FN-RMS and compare it to oncosuppressor miRNAs (hsa-miR-449a-5p and hsa-miR-340-5p).
  • MPC mitochondrial pyruvate carrier
  • Human FN-RMS (RD18 and RD) and murine FN-RMS (KMR46) were used as models, and the treatment effects were investigated via immunofluorescence staining, quantitative real-time polymerase chain reaction (RT-qPCR), and western blot in order to assess changes at both the morphological and molecular levels.
  • RT-qPCR quantitative real-time polymerase chain reaction
  • EVs extracellular vesicles
  • MPC inhibition led to a decrease in cell proliferation and formation of multinucleated structures, through the alteration of tumour metabolism by MPC inhibition.
  • miRNA treatment led to cell death and the formation of large multinucleated structures.
  • Both treatments showed an increase in the fusion percentage, i.e., increase in the amount of multiple nuclei cells, and with the feature of enlargement of cells after miRNA treatment, the results indicate that these cells are senescent-like cells after treatment.
  • MPCi MPC inhibitor
  • the MPCi showed similar effects as the oncosuppressor miRNAs in terms of reduction in cell proliferation in human and murine FN-RMS. These results allow development of therapies such as the in vivo delivery of miRNAs through the use of EVs.
  • Mir-340 and/or mir-449a for use in the treatment or prevention of a tumour according to statement 3, in a human individual. 5. Mir-340 and/or mir-449a for use in the treatment or prevention of a tumour according to statement 3 or 4, wherein the tumour is a soft tissue sarcoma, such as a paediatric soft tissue sarcoma.
  • Mir-340 and/or mir-449a for use in the treatment or prevention of a tumour according to any one of statements 3 to 5, wherein the tumour is rhabdomyosarcoma.
  • rhabdomyosarcoma RMS
  • the rhabdomyosarcoma is selected from the group of embryonal RMS, alveolar RMS, spindle cell RMS, mixed-type RMS, pleomorphic RMS, and RMS with ganglionic differentiation and alveolar RMS.
  • rhabdomyosarcoma rhabdomyosarcoma
  • FP-RMS fusion-positive RMS
  • FN- RMS fusion-negative RMS
  • Mir-340 and/or mir-449a for use in the treatment or prevention of a tumour according to any one of statements 3 to 6, wherein mir-340 is hsa-mir340-5p and mir-449a is hsa-mir-449-5p.
  • a pharmaceutical composition comprising mir-340 and/or mir-449a.
  • composition according to statement 10 wherein mir-340 is hsa-mir340-5p and mir-449a is hsa-mir-449-5p.
  • a method of treating tumour in an individual comprising the step of administering mir-340 and/or mir-449a to said individual, thereby converting tumour or tumorigenic cells into cell with a non-tumorigenic phenotype.
  • mir-340 is hsa-mir340-5p and mir-449a is hsa-mir-449-5p.
  • Figure 1 Differences between different samples of KMR46 day 3 and day 5 at lOx magnification
  • A Graphical representation of KMR46 day 3 and day 5 nuclei counting for different sample groups.
  • B Graphical representation of KMR46 day 3 and day 5 nuclei size (pm) of different sample groups.
  • Figure 2 Differences between different samples of human FN-RMS day3 and day 5 at lOx magnification
  • A Graphical representation of human FN-RMS day 3 and day 5 nuclei counting for different sample groups.
  • B Graphical representation of human FN-RMS day 3 and day 5 nuclei size (pm) of different sample groups.
  • Day 3 Changes in protein contents after treatment with MPCi and miRNA, compared to the DMSO control group
  • FIG. 6 In vitro and in vivo effects of miR-449a+340 in FN-RMS.
  • FIG. 7 miRNA effects on metabolism.
  • B Downregulated Gene Ontology pathways in FN-RMS patients compared to skeletal muscle;
  • FIG. 8 MPC inhibition and oncosuppressor miRNAs similarly affect oxygen consumption rate, cell cycle and proliferation.
  • B Seahorse measurement of oxygen consumption rate (n>4);
  • D Nuclei counting,
  • E Size, and
  • F Fusion percentage in human FN-RMS. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • (n 3)
  • MPCi and miRNAs similarly target in vivo migration and proliferation, and improve overall exercise capability in mice.
  • FIG. 10 Epigenetic effects of miR-449a+340 on FN-RMS.
  • 2-DG 2-deoxyglucose ;
  • AGO Argonaute protein;
  • AKT Protein kinase B;
  • ALK Anaplastic lymphoma kinase;
  • Alpha-SMA Alpha smooth muscle actin;
  • ARMS Alveolar rhabdomyosarcoma;
  • ATP Adenosine triphosphate;
  • BCOR BCL6 corepressor;
  • BMPs Bone morphogenetic proteins;
  • BSA Bovine serum albumin;
  • CCND2 Cyclin- dependent kinase cyclin D2 (CCND2);
  • CPT1A Carnitine palmitoyltransferase ;
  • CTNNB1 Catenin beta 1;
  • CXCR4 C-X-C chemokine receptor type 4;
  • DMEM Dulbecco's Modified Eagle Medium;
  • DMEM-HG High glucose Dulbecco's
  • Rhabdomyosarcoma is a soft tissue sarcoma with characteristics of skeletal muscle lineage, having high incidence in young adults below the age of 20.
  • RMS Rhabdomyosarcoma
  • FP-RMS fusion-positive RMS
  • FN-RMS fusion-negative RMS
  • RMS can be subdivided into six histological groups, i.e. embryonal, alveolar, spindle cell, mixed-type, pleomorphic, and RMS with ganglionic differentiation.
  • ERMS embryonal RMS
  • RMS alveolar RMS
  • ERMS embryonal RMS
  • RMS alveolar RMS
  • FN-RMS typically have a high rate of single-nucleotide alterations, and the recurrent mutation typically occurs in cancer genes such as harvey-ras (HRAS), neuroblastoma-ras (NRAS), kirsten-ras (KRAS), anaplastic lymphoma kinase (ALK), fibroblast growth factor receptor 4 (FGFR4), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), f-box and wd repeat domain containing 7 (FBXW7), neurofibromatosis type 1 (NF1), TP53, catenin beta 1 (CTNNB1) or BCL6 corepressor (BCOR).
  • HRAS harvey-ras
  • NRAS neuroblastoma-ras
  • KRAS kirsten-ras
  • ALK fibroblast growth factor receptor 4
  • PIK3CA phosphatidyl
  • FN-RMS tumours are characterized by the loss of function TP53 mutations, and this is due to focal amplification of the mouse double minute 2 homolog (MDM2), a TP53 negative regulator.
  • PAX3-FOXO1 has the ability to alter the transcription of genes such as FGFR4 and insulin-like growth factor 2 (IGF-2).
  • IGF-2 overexpression occurs in FN-RMS due to loss of heterozygosity (LOH) at the llpl5.5 locus.
  • FN-RMS mutations occur in genes that affect the activation of the receptor tyrosine kinase (RTK)/RAS/phosphatidylinositol-3- kinase (PI3K) signaling axis, and somatic mutations occur in cell cycle genes, including CTNNB1, FBXW7, BCOR and p53, which commonly occur with the gain of chromosomes 2,8 and 13.
  • RTK receptor tyrosine kinase
  • RTK receptor tyrosine kinase
  • PI3K phosphatidylinositol-3- kinase
  • FP-RMS The characteristics of FP-RMS are sustained activation of the RTK/RAS/PI3K signaling axis, tumours with a high copy number of the N-MYC gene (due to the PAX3-FOXO1 fusion protein regulating the oncogenic transcription of N-MYC) and a loss of imprinting at the llpl5.5 locus which leads to IGF-2 overexpression. Therefore, inhibiting the expression and/or activity of PAX3-FOXO1 by using specific inhibitors is one of the main goals of current studies for treatment of FP-RMS.
  • FN-RMS fusion-negative RMS
  • RMS cells express PAX3, PAX7 and myogenic regulatory transcription factors MRFs).
  • PAX3 and PAX7 indicate both muscle-forming and neural crest cells.
  • MSCs mesenchymal stem cells
  • FN-RMS low-passage myoblasts
  • sarcomas from both low-passage MSCs and myoblasts.
  • MRFs myogenic factor 5 (MYF5) and myoblast determination protein (MyoD) [Clark et al. (1991) Br. J. Cancer 64(6), 1039-1042].
  • MYF5 and MyoD have important roles in regulating growth, proliferation and TPC activity in RMS. Not only is MYF5 a marker of TPCs in the zebrafish FN-RMS model, but also in vivo it can transmit tumour propagating potential to differentiated FN-RMS cells. Both MYF5 and MyoD are essential for RMS proliferation where together they regulate molecular programs that are commonly found in healthy muscle development and regeneration.
  • MYF5 and MyoD are located on the promoter and enhancer regions of certain genes, such as cyclin-dependent kinase cyclin D2 (CCND2), myogenin (MYOG), and cadherin 15 (m-cadherin, CDH15'). These genes regulate cell cycle progression and muscle differentiation. This is also the reason why when MYH5 and MyoD are knocked down, the aforementioned genes are commonly downregulated. In general, MyoD and MYF5 have essential roles in muscle cell identity regulation and cell cycle regulation. Both are needed for tumour growth and may regulate stem cell self-renewal in healthy muscles.
  • MPC Mitochondrial pyruvate carrier
  • the blockade of MPC activity promotes cytosolic accumulation of pyruvate which stops the intracellular production of pyruvate through lactate conversion and prevents the uptake and consumption of extracellular lactate by cancer cells. Therefore, when lactate was the only available nutrient, the blockage of MPC activity prevented cancer cell growth. Also, the MPC inhibition could force cells to go from partially aerobic glucose metabolism to strict anaerobic glycolysis which could also cause cytotoxic effects. Finally, when blocking lactate and glucose from entering the TCA cycle as fuel, the inhibition of MPC activity also massively decreases the oxygen consumption and thus sensitizes tumours to radiotherapy via local reoxygenation. However, the blockade of MPC activity leads to the inhibition of lactate uptake, but also alters glucose oxidation.
  • MPC activity could lead to aerobic glycolysis and induce growth of tumour cells.
  • a lower expression of MPC proteins has been shown to lead to poor survival rates in patients with different types of cancers, such as colon cancer, prostate cancer, and oesophageal cancer.
  • cancer cells undergo metabolic status changes where cells adapt to prevent cell death and to sustain continuous cell division.
  • One important characteristic of cancer metabolism is that tumour cells tend to metabolize glucose anaerobically, even in aerobic conditions. Compare to the aerobic glycolysis, this type of metabolism leads to increase in glucose uptake and production of lactate via fermentation of glucose..
  • This type of molecular adaptation is called the Warburg effect where glucose-derived carbon skeletons are used for macromolecule biosynthesis instead of oxidation via the TCA cycle in the mitochondria.
  • cancer cells alternatively use the glucose backbone to produce ribose and nicotinamide adenine dinucleotide phosphate (NADPH) for nucleotide synthesis, lipid biosynthesis and the maintenance of the cells' redox state.
  • NADPH nicotinamide adenine dinucleotide phosphate
  • Glutamine is a non- essential amino acid precursor, a substrate for gluconeogenesis, and also an alternative energy source for rapidly dividing cells.
  • FAS fatty acid synthase
  • PK pyruvate kinase
  • Rh30 cells the balance between energy production and the biosynthetic precursor production may be regulated by an activation of anaplerotic carboxylation and the malate/aspartate shuttle which promotes nicotinamide adenine dinucleotide hydrogen (NADH) hydride to migrate from the cytoplasm to the mitochondrion for oxidative phosphorylation.
  • NADH nicotinamide adenine dinucleotide hydrogen
  • PAX3-FOXO1 coordinates the expression of several PAX3 downstream genes, such as hepatocyte growth factor receptor (HGFR or c-MET), FGFR4, IGF-2 and C-X-C chemokine receptor type 4 CXCR4), which are key genes involved in the increase in tumour aggressiveness and metastasis recurrence of FP-RMS.
  • PAX3-F0X01 regulates the transcription of the GLUT4 gene which leads to an increase of glucose consumption in FP-RMS cells.
  • PAX3-FOXO1 also causes the downregulation of the phosphate and tensin homolog deleted on chromosome 10 PTEN) gene [101], thus resulting in the activity alteration of the PI3K/(protein kinase B)AKT/mammalian target of the rapamycin (mTOR) pathway).
  • acyl carnitines which facilitate lipid degradation. This could be the source of energy for FP-RMS cells for migration and metastasis.
  • Both FN-RMS and FP-RMS are related to the activation of RTK-dependent pathways, which include the PI3K/AKT and RAS pathways.
  • the PI3K/AKT pathway triggers the consumption of glucose, amino acids and other nutrients.
  • mTOR glycolysis and lactate production, lipogenesis and other protein synthesis are promoted.
  • HIF Hypoxia inducible factors
  • IL-8 proangiogenic interleukin-8
  • CXCR1 and CXCR2 7-transmembrane G-protein-coupled receptors
  • p53 has a role in inhibiting the expression of glucose transporters (GLUTs), for example GLUT1 and GLUT3/GLUT4, and it reduces glucose consumption in the glycolytic pathway.
  • GLUTs glucose transporters
  • PTEN which negatively regulates glycolysis and the PI3K/AKT/mTOR and HIF pathways. Therefore, for cells with no p53 activity, typically high sugar transport across the cell membrane and high glucose consumption are found. The chances to have a loss of p53 activity are higher in FN-RMS than in FP- RMS, with an incidence rate of 5-19%.
  • glucose consumption is higher since the expression of the GLUT1 and GLUT4 increase due to the loss of p53 activity. Additionally, increasing the expression of GLUT1 transporter increases glucose consumption and glycolytic use which eventually is the reason for high cell proliferation and invasiveness.
  • Activation of the RAS pathway is closely related to FN-RMS aggressiveness.
  • RAS-positive FN-RMS cells are related to increased oxidative stress.
  • FN-RMS tumours are found to have a high rate of G to T transversions due to oxidative damage.
  • FN-RMS promote methylation of certain genes which regulate metabolism, mitochondrial function and oxidative stress.
  • ROS mitochondrial reactive oxygen species
  • MAPK mitogen activated protein kinase
  • FN-RMS tumours could benefit from increased ROS levels, but at the same time, increasing accumulation of ROS-induced gene mutations could lead to cell death as well. Therefore, oxidative stress inducer, actinomycin-D could be the key to control tumour growth.
  • MicroRNAs belong to the class of non-coding RNAs (ncRNAs), with an average size of 22 nucleotides and having roles in regulating gene expression. Most miRNAs are first transcribed from DNA into primary miRNAs (pri-miRNAs), then inside the nucleus pri-miRNA is further processed into a precursor miRNA (pre-miRNA) by a class 2 RNase III enzyme called Drosha. After this, exportin-5 (EXP-5) helps with the transportation of pre-miRNA to the cytoplasm via nuclear pore complexes, a large proteinaceous channel located in the nuclear membrane. Inside the cytoplasm, pre- miRNAs are processed by Dicer to become mature miRNAs.
  • Dicer an RNase III type protein
  • Dicer-1 is responsible for miRNA maturation
  • Dicer-2 is responsible for siRNA maturation.
  • AGO Argonaute
  • RISC effector RNA-induced silencing complex
  • miRNAs interact with the target mRNAs via the 3' untranslated region (3' UTR), or in some cases, via other sites such as the 5' UTR, a coding sequence, and gene promoters. Depending on the situation, miRNAs can activate translation or regulate transcription as well. However, interaction of miRNAs with target genes can be affected by many factors, for example, the subcellular location of miRNAs, the abundancy of miRNAs and mRNAs and the affinity of their interactions. miRNAs secreted into extracellular fluids can be transported to target cells by vesicles. Extracellular miRNAs act as chemical messengers which can mediate cell-cell communications.
  • the eIF4F complex is related to the initiation of translation.
  • This complex consists of subunits, such as eIF4A, eIF4E, and eIF4G.
  • the eIF4E subunit recognizes the mRNA 5' terminal cap and starts the initiation process.
  • another initiation factor, eIF3 reacts with eIF4G and helps with the 40S ribosomal subunit assembly at the 5'end of the mRNA.
  • the elongation process is then initiated by the 60S ribosomal subunit and 40S preinitiation complex binding at the AUG codon of the mRNA.
  • miRNA-induced silencing complex may repress the elongation process, and it could promote early ribosome dissociation from mRNAs.
  • miRISC miRNA-induced silencing complex
  • miRISC prevents the mRNA from turning into a circular shape which causes translational inhibition.
  • miRISC can inhibit the 60S ribosomal subunit from joining with the 40S subunit, leading to translational repression.
  • miRNA or miRISC can have an effect on translational repression due to the increase in target mRNAs in the processing bodies.
  • miRNAs The degradation of target mRNAs is induced by the AGO protein slicer activity. miRNAs also affect this process. Some other mechanisms are also involved, such as deadenylation, decapping and exonucleolytic digestion of mRNA. However, the number, type, and position of mismatches in miRNA and mRNA duplexes are essential for the selection of the degradation or translational repression mechanisms. miRNAs and myogenesis
  • MRFs Muscle development is regulated by the MRFs: MYF5, MyoD, MYOG and MRF4 which promote the formation of muscle fibres.
  • miRNAs in this case have an important role in the development of skeletal muscle tissues [Buckingham (2001) Curr Opin Genet Dev 11, 440-448].
  • some typically upregulated miRNAs such as miR-133a-l, miR-133a-2, miR-133b and miR-206, have an important role in myogenic differentiation [Chen et al (2006) Nat Genet 38, 228-233; Kim et al. (2006) J Cell Biol 174, 677-687].
  • miRNAs in muscle tissue can be subdivided into two types, muscle-specific myo- miRNAs and non-myomiRNAs which are found in both skeletal and cardiac muscles [Gasparini et al. (2019) Int J Mol Sci 20, 5818].
  • muscle-specific myo-miRNAs can be either uniformly expressed over muscles (i.e., miR-1 and miR-133a), or can be highly expressed in slow-twitch/type I muscles (i.e., miR-206, miR-208b and miR-499).
  • Myo-miRNAs correspond to only 3% of expressed miRNAs in quiescent satellite cells, but in differentiated cells myo-miRNAs correspond to 74% of expressed miRNAs.
  • regulation is needed to activate specific miRNAs at different stages of myogenesis to achieve optimal balance between proliferation and myoblast differentiation.
  • miRNAs as a biomarker and the role in RMS development miRNAs can regulate post-transcription of different phases of muscle-specific processes, which shows their ability as biomarkers in the development of RMS [Bersani et al. (2016) cited above].
  • the myo-miRNAs are the key to skeletal muscle tissue differentiation, where their downregulation is related to the dedifferentiation of the phenotype in RMS [Novak et al (2013) Pediatr Blood Cancer 60, 1739-1746].
  • the overexpression of miR-1 in RD cell lines regulates the gene expression of muscle tissue and cell cycle arrest, whereas miR-133a decreases the expression of muscle markers.
  • miR-1, miR-22 and miR-206 are downregulated while miR-29 is deregulated in a few FP-RMS [Bersani et al. (2016) Cancer Res 76, 6095-6106]) .
  • miRNAs have important effects on the development and progression of RMS, these molecules could be interesting as therapeutic targets in pediatric tumours.
  • overexpression of miR-378 can cause changes in apoptosis, migration, and viability.
  • miR-183 has an oncogenic role and it targets two tumour suppressor genes, specifically early growth response protein 1 EGR1) and PTEN.
  • EGR1 early growth response protein 1
  • Extracellular vesicles are mediators of cell-cell communication and play an important role in cancer metastasis. Therefore, their use as biomarkers for clinical purposes is being explored extensively .
  • EVs are lipid bilayer vesicles with a diameter size of 40-1000 nm which are produced and released by cells or detached from the plasma membrane. EVs can be divided into subtypes based on their biogenesis, release pathway, size, content, and function. The three main subtypes of EVs are microvesicles (MVs), exosomes, and apoptotic bodies.
  • Exosomes or intraluminal vesicles have a single outer membrane and are secreted by all types of cells. Exosomes can be found in many fluids such as plasma, urine, saliva, or tears. Exosomes have a diameter size of 30-150 nm and are formed via an endosomal route. Early endosomes formed from inward budding of the cells' plasma membrane, later mature into multivesicular bodies (MVBs) which are involved in protein sorting, recycling, storage, transport, and release. Then, MVBs are either degraded by the lysosomes or fuse with the cells' plasma membrane to secrete exosomes and other contents into the extracellular space. The fate of a MVB depends on the level of cholesterol it contains.
  • Vesicles rich in cholesterol are secreted while vesicles with a low cholesterol level are degraded by the lysosomes.
  • the formation of MVBs is promoted by growth factors.
  • the cell adapts its production of exosomes based on its needs.
  • exosomal formation and MVB transportation are regulated by endosomal sorting complexes required for transport (ESCRT) proteins and with accessory proteins. These are commonly considered as “exosomal marker proteins”. These "marker proteins” can be found in all exosomes, independent from cell type.
  • the transmembrane proteins CD63, CD9 and CD81 belong to the tetraspanin family, which are also often found in exosomes.
  • exosomes The biological purpose of exosomes includes cell-cell communication, cell maintenance and tumour progression. Furthermore, when exosomes act as antigen- presenting vesicles, immune responses can be stimulated. Exosomes in the nervous system can promote myelin formation, neurite growth, and neuronal survival, which in the end also help with tissue repair and regeneration.
  • exosomes are also suitable for immunological applications since they have a relatively long circulating half-life, are tolerated by the human body, are able to penetrate cellular membranes, and have the potential to target specific cell types.
  • exosomes One of the important properties of exosomes is its inherent property, which can act inherently as antigen presenting vesicles, and makes exosomes ideal to use as a drug delivery system. Nucleic acids and proteins can be introduced into exosomes which can be further modified to target specific cells.
  • oncosuppressor microRNAs (miR-449a+miR-340) was identified, which showed downregulation of MPCI and MPC2 upon treatment of FN-RMS models with these miRNAs.
  • RMS is a type of STS, and the most common type in children. RMS tumours are histologically similar to normal foetal skeletal muscle. As mentioned, there are two major subtypes of RMS: FP-RMS and FN-RMS. Looking at the morphology, FP-RMS typically are small round densely packed cells, whereas FN-RMS typically consist of spindle-shaped cells and are rich in stroma.
  • MPCi mitochondrial pyruvate carrier inhibitor
  • MPCi- and miRNA-treated groups For both murine KMR46 and human RD18 FN-RMS cells, a decrease in the confluency was found in the MPCi- and miRNA-treated groups. Furthermore, the MPCi-treated group showed elongation of cells and intention to form multinucleated-like structures, whereas in the miRNA-treated group, there were formations of gigantic mono-, bi-, tri- or multinucleated cells instead more visible in murine FN-RMS. This could be due to the senescence effects, which led to a lower cell confluency and formation of larger cells. From these results, a possible preliminary conclusion could be that the MPC inhibition has effects of inhibiting tumour growth or proliferation, by altering cancer metabolism.
  • Literature shows that the decrease in MPC activity can lead to aerobic glycolysis and induce the growth of tumour cells, metastasis, and poor survival of patients with different types of cancers, such as colon cancer, prostate cancer, and esophageal cancer.
  • an planation for MPCi having possible anti-cancer effects instead of pro-cancer effects is that the role of MPC is highly dependent on the tissue type and metabolic inclinations of cancers. Since colon, prostate, and esophageal cancers arise from epithelial cells, whereas RMS can arise from mesenchymal stem cells or muscle progenitor cells, there might be an effect on the response of MPC inhibition.
  • cancers that use mitochondrial pyruvate for tumour growth and glutamine for glutathione production are typically negatively affected by MPCi.
  • RMS tumours express myogenic regulatory transcription factors, such as MYF5 and MyoD, which have important roles in regulating growth, proliferation and tumour propagating cell activity in RMS. Furthermore, the binding sites of MYF5 and MyoD are located on the promoter and enhancer regions of some genes, such as MYOG, which regulate cell cycle and muscle differentiation. Indeed, the effect of MPCi treatments on both KMR46 and human FN-RMS samples affected MYF5/ Myf5, MyoD, and MYOG/ Myog expressions as indicated by the RT-qPCR analysis. In the KMR46 samples, after MPCi treatment, Myf5 was upregulated whereas MyoD and Myog were downregulated.
  • MYF5 and MyoD myogenic regulatory transcription factors
  • MYF5 was undetermined, MyoD was upregulated, and MYOG was again downregulated.
  • MYOG was again downregulated.
  • MYF5 and MyoD share the same binding site but due to functional divergence, each established a separate muscle lineage. Therefore, regulation of either MYF5 or MyoD could lead to changes in skeletal muscle differentiation.
  • regulation of MRFs due to MPC inhibition may promote cells to differentiate into skeletal muscle-like cells whereas the miRNA-treated cells are more likely to differentiate into smooth muscle-like cells.
  • MPCi treatment can be that it saves the cells from being trapped in the MYF5 path, by regulating the expression of MYF5 and MyoD. Thus, the cells either go down the same path or by another path, which eventually leads to a more successful myogenesis.
  • the tumour microenvironment consists, besides tumour cells, of fibroblasts, endothelial cells, immune cells, and non-cellular components (including exosomes and cytokines) which form an internal environment where tumour cells survive.
  • the TME plays an important role in all stages of tumourigenesis.
  • miRNAs can be carried by exosomes, which have an important role in intercellular transport and signalling.
  • the heterogeneity of the TME is mainly due to miRNAs' activation of cancer-associated fibroblasts which also reshape the extracellular matrix (ECM) and thus promote the spreading of tumour cells. Therefore, using extracellular vesicles (EVs) or exosomes as carriers for oncosuppressor miRNAs could be an interesting way to alter the proliferation and metabolism of tumour cells.
  • ECM extracellular matrix
  • Human RD18 and murine KMR46 FN-RMS cell lines were cultured in high glucose Dulbecco's Modified Eagles Medium (DMEM-HG) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (P/S). Cells were maintained under standard incubator conditions humidified atmosphere (95% air, 5% CO2, 37°C) and passaged twice weekly with 0.25% Trypsin-EDTA.
  • DMEM-HG high glucose Dulbecco's Modified Eagles Medium
  • FBS fetal bovine serum
  • P/S Penicillin/Streptomycin
  • cells were seeded at 1 :20 KMR46/human MABs ratio on collagen-coated vessels in IMDM 15% FBS medium, 1% L-glutamine, 1% non- essential amino acids, 1% sodium pyruvate, 1% ITS, 1 : 10,000 basic FGF, 0.2% betamercaptoethanol. After 24 h, medium was changed and OptiMEM with miRNA- lipofectamine or lipofectamine was added to the wells. A total of 24 h after miRNA addition, cells were differentiated in DMEM 2% horse serum medium for 96-120 h in 5% 02/5% CO2 at 37°C.
  • Cells were transfected with lentiviral particles expressing EFla-eGFP-P2A-fLuc and selected with 1 pg/mL puromycin for 2 weeks.
  • GFP was used as a standard gene expression tracer in vitro.
  • Firefly luciferase (fLuc) was used as an optical reporter gene, upon coelenterazine administration, to detect the cell line engraftment in vivo via IVIS bioluminescence imaging (BLI).
  • cells were seeded at 12-15.000 or 50.000 cell number in 24- wells or 6-wells, respectively.
  • cells were transfected using 3 pl/mL Lipofectamine 2000 and 1.6 pl/mL of hsa-miR-449a-5p/-340-5p, or hsa-miR- 181a-5p/-212-3p (all MISSION® microRNA Mimics, Sigma-Aldrich), at 10 pM concentration resuspended in OptiMEM-medium.
  • MPCi treatment cells were treated with 8 pl (final concentration is 40 pM) of Mitochondrial pyruvate carrier inhibitor (UK-5099, Sigma-Aldrich) in 2 ml of Opti-MEMTM I Reduced Serum Medium.
  • DMSO Dimethyl sulfoxide
  • mouse anti-MyHC (DSHB #MF20), 1 :3; rabbit antilamin A/C (Epitomics #2966-1), 1:600; mouse anti-Myogenin (Invitrogen #AB_10977211), 1: 100; rabbit anti-MyoD (Thermo Fisher Scientific #MA5-12902), 1: 100.
  • Imaging was performed at Eclipse Ti microscope (Nikon) by means of Image- Pro Plus 6.0 software (Nikon).
  • 20,000 pretreated cells were seeded into 6-well Ultra-Low Attachment plates (Corning, USA) in serum-free DMEM/F12 (Invitrogen, USA), supplemented with B27 (lx, Invitrogen, USA), 20 ng/ml EGF (Peprotech, USA), 20 ng/ml bFGF (Invitrogen), and 1% Penicillin/Streptomycin (Sigma, USA). Pictures were taken every day for 11 days.
  • Seahorse XFp Extracellular Flux Analyzer was employed to measure oxygen consumption rates (OCR) using the Mito Stress Test Kit following the manufacturer's protocol. Briefly, pretreated 40,000 cells/well were plated Seahorse XFp cartridge for 4 hours in growth medium. Following attachment to the well, medium was changed with XF media pH 7.4 supplemented with 2mM L-Glutamine, 10 mM glucose and 1 mM sodium pyruvate. Basal OCR was measured for 4 cycles, then 8 pM oligomycin was injected to inhibit ATP-linked respiration, followed by 4.5 pM FCCP to measure maximal respiration, and finally 10 pM rotenone/antimycin A was injected to completely inhibit all mitochondrial respiration. After each experiment, protein concentration was measured and wells were normalized using the Wave software.
  • EdU staining was performed using Click-iT® EdU Alexa Fluor® 647 Flow Cytometry Assay according to manufacturer's advice. Briefly, after EdU incubation of l-2h, cell pellets were fixed with 4% PFA, permeabilized with 0.5% Triton X-100 in PBS for 20 min, and subsequently treated with Click-iT reaction cocktail at room temperature for 10 minutes. Subsequently, cells were washed and incubated in propidium iodide (PI) staining buffer (20 pg ml-1 PI, 0.2 mg ml-1 RNase A, PBS) for 30 min at room temperature. Cell cycle distribution was analysed by flow cytometry (BD FACSCanto HTS) and FlowJo software.
  • PI propidium iodide
  • RNA-sequencing libraries were constructed with the Lexogen library. Samples were indexed with unique adapters and pooled for single read (50 bp) sequencing in Illumina HiSeq2000. RNA-seq reads were aligned with TopHat v2.0.2 to the mouse genome mmlO. Transcripts were assessed and quantities were determined by Cufflinks38. Differential expression levels were assessed using DESeq2.
  • Sequencing reads were trimmed and aligned to assembly GRCh38 using bwa mem. Duplicate reads and reads mapping to mitochondrial sequences were subsequently removed. Chromatin accessibility peaks were called using MACS2 and annotated using HOMER and/or ChipSeeker. Differential accessibility analysis was performed using the R/Bioconductor package DiffBind.
  • mice C57/BI6 mice were injected in the femoral artery with either 1 x 105 KMR46 Fluc+ cells (untreated group) or with 1 x 105 KMR46 Fluc+ cells pretreated for 3 days with s-PMC (treated group).
  • cells were suspended in 50 pl saline water.
  • the mice were monitored through BLI every other day starting from day 7 after tumour injection for 10 days.
  • mice were placed in the flow chamber of IVIS® Spectrum. Subsequently, 126 mg/kg of D-luciferin was injected subcutaneousl. Hence, consecutive frames were acquired until the maximum signal intensity was reached. Pulse/sec intensities were calculated by comparing the same ROI for all the animals, after subtracting the background signal coming from not injected mice.
  • Sample size for in vitro/in vivo experiments was calculated by means of Sample Size Calculator (parameters: power, 0.80; alpha, 0.05). When applicable, sample size analysis was based on average values obtained from preliminary optimization/validation trials. Depending on the experiment, two-tailed unpaired Student's T-tests analysis or two-way ANOVA test was performed. All statistical analyses were conducted using Prism v9.1.0 (GraphPad).
  • RD and RD18 Human FN-RMS (RD and RD18) and murine FN-RMS (KMR46) cell lines were used.
  • RD and RD18 are different cell lines but both are human FN-RMS with characteristics of expressing MYOG, p21 and Desmin.
  • KMR46 is a cell line derived from the mouse model MyoD-cre x KRAS G12D x TP53F/-).
  • the KMR46 cell line has similar characteristics to FN- RMS patients, which indicates it is a suitable model to use for this research.
  • RD18/RD and murine KMR46 FN-RMS cell lines were cultured in high glucose Dulbecco's Modified Eagle Medium (DMEM-HG) (GibcoTM, Thermo ScientificTM, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (P/S) (GibcoTM). The medium was filtered with 0.22
  • DMEM-HG high glucose Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • P/S Penicillin/Streptomycin
  • MPCi Mitochondrial pyruvate carrier inhibitor
  • DMSO Dimethyl sulfoxide
  • a Nikon Eclipse Ti-S microscope (Nikon, Minato, Tokyo, Japan) was used and the collected images were further processed with the Image J software (National Institutes of Health (NIH, Bethesda, MD, USA).
  • RT-qPCR Quantitative real-time polymerase chain reaction
  • RNA/supernatant was collected.
  • RNA collected after the DNase I treatment was then used for cDNA synthesis, by using a cDNA Reverse Transcription Kit (High-Capacity cDNA Reverse Transcription Kit, Applied BiosystemsTM).
  • a cDNA Reverse Transcription Kit High-Capacity cDNA Reverse Transcription Kit, Applied BiosystemsTM.
  • 1 pg of RNA was utilized and reverse- transcribed using 10 pl of cDNA synthesis buffer, 2 pl of reverse transcriptase and a total of 8 pl of sample RNA and DEPC-treated water.
  • the synthesis of cDNA was done in a thermocycler (Biometra T3000 Thermocycler, Biometra, Analytik Jena, Jena, Germany).
  • 1 pl of E. coli RNase H was added to each sample and placed back into the thermocycler.
  • SNAI1 PTEN, MYLK, MYBL2 and MEF2C. All primers were synthesized and purchased from Integrated DNA Technologies (IDT, Coralville, IA, USA).
  • pellets were lysed in either 60 pl or 90 pl of RIPA buffer (Thermo ScientificTM, #89900).
  • the solutions were sonicated twice for 10 seconds at an amplitude of 10% (Sonifier® Cell Disruptor, Branson Ultrasonics Corporation, Danbury, CT, USA), incubated at room temperature for 10 minutes and centrifuged at 16000 rpm, for 15 minutes at 4°C.
  • the supernatants were collected and quantitatively measured by using the NanoDrop spectrophotometer (NanoDrop® ND-1000, Isogen life science B.V., De Meern, NL).
  • NanoDrop® ND-1000 Isogen life science B.V., De Meern, NL
  • the gel was run at 120V (PowerPacTM Basic Power Supply, Bio-Rad Laboratories, Inc.) until the dye front was close to the end of the gel. Then, the proteins were electro-transferred onto a nitrocellulose membrane with transfer buffer, at 200 amperes for 2 hours and 30 minutes. All of the equipment was purchased from Bio-Rad Laboratories, Inc. (Mini-PROTEAN Tetra Cell). The membranes were then stained with Ponceau (Sigma- Aldrich), to visualize proteins and to check if the transfer was successful.
  • Ponceau Sigma- Aldrich
  • the membranes were washed with TBS-Tween (Thermo ScientificTM) and were blocked with 5% (w/v) skim milk (Sigma-Aldrich) in TBS-Tween for one hour at room temperature. After being washed with TBS-Tween twice for 10 minutes on the shaker, the membranes were incubated with primary antibody in 2.5% (w/v) skim milk in TBS-Tween overnight at 4°C. On the second day, the primary antibodies were recollected, and then membranes were washed 3 times with TBS-Tween for 5 minutes. Membranes were incubated with secondary antibody in 2.5% (w/v) skim milk in TBS- Tween for one hour at room temperature.
  • the membranes were incubated with (1:3300 - in 2.5% (w/v) milk in TBS-Tween) anti-rabbit secondary antibody (Abeam, ab6721) (Cell Signaling Technology, #7074) and (1:3300 - in 2.5% (w/v) milk in TBS-Tween) and anti-mouse secondary antibody (Abeam, ab97046).
  • EVs were extracted from the LHCN-M2 (human immortalized myoblasts) cells.
  • cells were cultured in medium (1 vol. 199 medium, 4 vols. DMEM, 20% FBS and 50
  • the cells were left in the differentiation medium (DMEM, 10 pg/ml insulin, and 50 pg/ml gentamycin) for 4 days. Finally, the medium was collected for EV extraction.
  • the collected medium was centrifuged at 500g for 10 minutes (Eppendorf Centrifuge, Model 5810, Eppendorf), 3000g for 20 minutes (Avanti®J-E high-speed centrifuge, Beckman Coulter life sciences, IN, USA), 12,000g for 20 minutes (Beckman coulter), and then 100,000g for 70 minutes (Optima L-90K Ultracentrifuge, Beckman Coulter). After removal of the supernatant, the pellets were resuspended in 5 ml of PBS, centrifuged again at 100,000g for 70 minutes. Finally, the supernatant was removed, and the pellets were resuspended in 400 pl of PBS and stored at -80°C. All of the centrifugation steps were done at 4°C.
  • PBS was used to dilute the isolated EVs to reach a final volume of 500 pl.
  • the suspension was filtered with a 0.2 pm filter (Corning® syringe filters, CLS431212, Corning).
  • 50 pl of lOx Exo-Red (ExoGlowTM - Protein EV labelling kit, System Biosciences, Palo Alto, CA) were added to the purified EVs, incubated at 37°C for 10 minutes and placed on ice for 30 minutes. Finally, the labelled EVs were placed in a 24-well plate.
  • PBS was used to dilute the isolated EVs with a dilution factor of 1: 5 (i.e., 400 pl of EVs in 1.6 ml of PBS), and the suspension was filtered with a 0.2 pm filter.
  • the purified EV samples were then loaded into the sample chamber of a Nanosight machine (NanoSight LM10, Malvern Panalytical Ltd, Malvern, UK) and the quantification and size measurement were done by using the Nanosight NTA 2.3 software (Malvern Panalytical Ltd).
  • the miRNA signature was determined of patients diagnosed with embryonal RMS. Data from Bersani et al. (2016) Cancer Res. 76, 6095-6106. (accession number ID PRJNA326118) were used to identify miRNAs not expressed in FN-RMS patients. The predicted genes targeted by the downregulated miRNAs were cross-checked and identified the pathways using g: Profiler. These pathways mainly involved include cell cycle, hippo signalling, fatty acid metabolism and central carbon metabolism. The combination of the key microRNAs that could be used as novel therapeutic agents was identified by checking the single pathways targeted by the microRNAs using DIANA software, and miR-449a and miR-340 were identified as the two key microRNAs in the downregulated microRNA pool.
  • Example 3.miR-449 and miR-340 dramatically affect tumourigenicity of FN- RMS
  • the role of the downregulated miRNAs in FN-RMS was investigated, by transfecting both human and mouse in vitro FN-RMS models with miR-449a and miR-340 and subsequently checking the perturbation effects at the transcriptome level with RNAseq analysis. Both the principal component analysis and a volcano plot showed a remarkable difference in miRNA-treated cells compared to vehicle-treated ones.
  • the downregulated genes are mainly involved in cell cycle, while among the top upregulated pathways in miRNA-treated samples cilium organization and cilium assembly were identified. This is in line with previously reported data regarding quiescent muscle cells that upon exit from the cell cycle undergo cilium formation.
  • oncosuppressor miRNAs were tested and the potential effects in vitro and in vivo were checked.
  • the proliferation level there was a reduction in proliferation in FN-RMS treated cells (figure 6A).
  • the effects on proliferation were checked in an in vitro hybrid system by co-culturing FN-RMS and mesoangioblasts (MABs) (Figure 6B). After transfection there was no tangible effect in terms of reduced number of cells in healthy skeletal muscle cells but solely in FN-RMS, which further confirmed the hypothesis of the specificity of these miRNAs in targeting the tumorigenic apparatus of FN-RMS.
  • RNA-treated tumour cells were treated using the miRNA combination, and subsequently injected the treated cells in C57BI/6 mice (Figure 6E). After 15 days, the reduction in proliferation of miRNA-treated tumour cells after a single treatment was still statistically significant compared to vehicle-treated implanted cells ( Figure 6F). Finally, as the main issue with respect to FN-RMS involves the metastasis to the lungs, the question arises whether RNA-treated tumour cells would have a reduced capacity at migrating compared to vehicle-treated ones. Indeed, both in vitro ( Figure 6G) and in vivo (Figure 6H) analysis showed a reduced migration potential of miRNA- treated cells.
  • Example 4 miR-449a+340 and mitochondrial pyruvate carrier (MPC) inhibition similarly affect proliferation and metastasis
  • MPCi mice performed significantly greater than the vehicle mice in terms of power on day 7, 9 and 13.
  • the power exerted was significantly greater than the one exerted by the vehicle mice on all days.
  • MPCi and miRNA treated tumour bearing mice were able to perform more similarly to sham mice, in contrast to the poor performance of vehicle mice, affected by the tumour.
  • MYOG reduced levels of MYOG were found both after miRNA and MPCi treatment ( Figure 9H). miRNA modulates and reverses the tumorigenic phenotype at the epigenetic level.
  • PBX1 was identified as the transcription factor with the highest number of enriched features ( Figure 5.7D-E).
  • snRNAseq single-nuclei RNAseq shows upregulation of myoblast feature CREB5, as well as senescence feature OXR1, in miRNA-treated FN- RMS
  • EZH2, TOP2A and MYBL2 were further investigated. These genes were found to be upregulated in FN-RMS patients compared to skeletal muscle, and they are downregulated by the miRNA combination in FN-RMS. the effects of drugs targeting these genes either alone or in combination were investigates.
  • Tazemetostat (EZH2 inhibitor), Doxorubicin (TOP2A inhibitor) and Topotecan (MYBL2 inhibitor) were determined using XTT proliferation assay which assesses cell viability as a function of redox potential.
  • Tazemetostat alone did not have effects, while at 96h Doxorubicin reduced the activity to 22.8% and Topotecan to 62.1% on FN-RMS cells compared to DMSO. As these targets are downregulated at the same time with miRNA treatment, the effect of these drugs combined was tested. Tazemetostat synergistically worked with Doxorubicin (18.9%) more than with Topotecan (42.3%) in reducing cell activity, while Doxorubicin combined with Topotecan has a dramatic effect on cell proliferation (2%). However, the synergy of the three drugs together has the most spectacular effect, with activity being reduced to 0.4% compared to vehicle-treated cells.
  • Example 6 MPCi-treated cells show similar effects as mi NA-treated samples when looking at the morphology of the cells
  • Example 7 MPCi-treated cells show similar effects as miRNA-treated samples in number, size, and multinucleation in the murine KMR46 cell lines When looking at the molecular level of different sample groups, three aspects were evaluated. First, the number of nuclei on day 3 and day 5 of different groups were compared. This was done to check if there was any effect on the proliferation rate of tumour cells after treatment with MPCi or miRNAs. The results showed that on day 3 there was already a decrease in the proliferation rate of the MPCi-treated group compared to the DMSO group. On day 5, the reduction in the rate of proliferation was even more significant (P ⁇ 0.0001) in the MPCi-treated samples.
  • Example 8 Western blot results show changes of MYF5 and MYOG protein contents after MPCi- and miRNA-treatment
  • exosomes isolated from the LHCN-M2 cells were visualized by using Exo-Red and checked under the microscope. The results show the presence of exosomal clusters at lOx magnification.
  • Nanosight was used for quantification of EVs and evaluation of the particle diameter.
  • the results showed EVs isolated from the LHCN-M2 cells had an average size of 143 +/- 92 nm, with a concentration of 0.14 +/- 0.13 E8 particles/ml (Figure 5).

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Abstract

The invention relates to the use of Mir-340 and mir-449a the treatment or prevention soft tissue sarcomas, such as rhabdomyosarcoma.

Description

TREATMENT OF RHABDOMYOSARCOMA
FIELD OF THE INVENTION
The invention relates to treatment of cancers with miRNAs.
BACKGROUND OF THE INVENTION
Rhabdomyosarcoma (RMS) occurs in 50% of paediatric soft tissue sarcomas and accounts for 3-4% of all paediatric cancers. The incidence is high below the age of 20, after which the incidence decreases to 1% of adult malignancies. In most cases, paediatric RMS tumours occur in the head and neck region.
When looking at the treatment for RMS, during the past 40 years there have been no significant changes, and the survival rate of metastatic and recurrent patients remains relatively the same at 21% and 30%, respectively. There is still plenty of space for improvement such as minimizing toxicities for chemotherapy and/or radiation therapy, both in short and long-term toxicity and especially for younger RMS patients. For metastatic RMS, proper and effective local therapy to treat specific regions is highly needed. To achieve these more targeted therapies, personalized and genotype-guided approaches are needed, as well as a better and effective treatment for the heterogeneity of RMS.
SUMMARY OF THE INVENTION
A combination of oncosuppressor microRNAs is identified and their effects on the tumorigenicity and the metabolism of pyruvate in FN-RMS were characterized.
The present invention investigates the effect of mitochondrial pyruvate carrier (MPC) inhibition in human and murine FN-RMS and compare it to oncosuppressor miRNAs (hsa-miR-449a-5p and hsa-miR-340-5p). Human FN-RMS (RD18 and RD) and murine FN-RMS (KMR46) were used as models, and the treatment effects were investigated via immunofluorescence staining, quantitative real-time polymerase chain reaction (RT-qPCR), and western blot in order to assess changes at both the morphological and molecular levels. To investigate the potential use of extracellular vesicles (EVs) as delivery vehicles of miRNAs, EVs were isolated from the LHCN-M2 cells and were investigated via exosome labelling and Nanosight analysis.
Herein, the effect of MPC inhibition on human and murine FN-RMS were compared to oncosuppressor miRNAs (hsa-miR-449a-5p and hsa-miR-340-5p). Through immunofluorescence staining, the results indicated that MPCi treatment led to a decrease in cell proliferation and formation of multinucleated structures, through the alteration of tumour metabolism by MPC inhibition. miRNA treatment led to cell death and the formation of large multinucleated structures. Both treatments showed an increase in the fusion percentage, i.e., increase in the amount of multiple nuclei cells, and with the feature of enlargement of cells after miRNA treatment, the results indicate that these cells are senescent-like cells after treatment. When looking at the gene expression after the treatment, there were several important genes downregulated after MPC inhibition, such as ACTAl/Actal, MYOG/Myog, TNNIl/Tnnil, TNNI2/Tnni2, TNNTl/Tnntl, and FGFR4/Fgfr4. Since RMS tumours express myogenic regulatory transcription factors, regulating some of these factors, might lead to the regulation of downstream MRFs and thus have an effect on the development of skeletal muscle. The effects of oncosuppressor miRNAs and the role of pyruvate metabolism on myogenic regulatory factors was determined, to investigate the in vivo delivery of miRNAs using EVs in human and murine FN-RMS.
Compared to the vehicle-treated group, the MPC inhibitor (MPCi) led to a decrease in cell proliferation and promoted cells to form multinucleated structures while miRNA treatment led to cell death and formation of enlarged multinucleated cells. In both treatments, an increase in the fusion of cells was also found. MPCi led to the downregulation of several important genes in the myogenic pathway, such as ACTA1, MYOD, MYOG, TNNI1, TNNI2, TNNT1, FGFR4. Through western blot analysis, reduced MYF5 and MYOG protein contents were observed which were not significantly different between treated and untreated samples. Finally, through EVs analysis, presence of exosomal clusters was found, and the average size and concentration of the isolated EVs were measured.
The MPCi showed similar effects as the oncosuppressor miRNAs in terms of reduction in cell proliferation in human and murine FN-RMS. These results allow development of therapies such as the in vivo delivery of miRNAs through the use of EVs.
The invention is further summarised in the following statements:
1. Mir-340 and/or mir-449a for use as a medicament.
2. Mir-340 and/or mir-449a for use as a medicament according to statement 1, wherein mir-340 is hsa-mir340-5p and mir-449a is hsa-mir-449-5p.
3. Mir-340 and/or mir-449a for use in the treatment or prevention of a tumour.
4. Mir-340 and/or mir-449a for use in the treatment or prevention of a tumour according to statement 3, in a human individual. 5. Mir-340 and/or mir-449a for use in the treatment or prevention of a tumour according to statement 3 or 4, wherein the tumour is a soft tissue sarcoma, such as a paediatric soft tissue sarcoma.
6. Mir-340 and/or mir-449a for use in the treatment or prevention of a tumour according to any one of statements 3 to 5, wherein the tumour is rhabdomyosarcoma.
7. Mir-340 and/or mir-449a for use in the treatment or prevention of rhabdomyosarcoma (RMS) according to statement 6, wherein the rhabdomyosarcoma is selected from the group of embryonal RMS, alveolar RMS, spindle cell RMS, mixed-type RMS, pleomorphic RMS, and RMS with ganglionic differentiation and alveolar RMS.
8. Mir-340 and/or mir-449a for use in the treatment or prevention of rhabdomyosarcoma (RMS) according to statement 6 or 7, wherein the rhabdomyosarcoma is fusion-positive RMS (FP-RMS) or fusion-negative RMS (FN- RMS).
9. Mir-340 and/or mir-449a for use in the treatment or prevention of a tumour according to any one of statements 3 to 6, wherein mir-340 is hsa-mir340-5p and mir-449a is hsa-mir-449-5p.
10. A pharmaceutical composition comprising mir-340 and/or mir-449a.
11. The composition according to statement 10, wherein mir-340 is hsa-mir340-5p and mir-449a is hsa-mir-449-5p.
12. A method of treating tumour in an individual comprising the step of administering mir-340 and/or mir-449a to said individual, thereby converting tumour or tumorigenic cells into cell with a non-tumorigenic phenotype.
13. The method according to statement 12, wherein the tumour is rhabdomyosarcoma.
14. The method according to statement 12, wherein mir-340 is hsa-mir340-5p and mir-449a is hsa-mir-449-5p.
DETAILED DESCRIPTION
Figure legends
Figure 1. Differences between different samples of KMR46 day 3 and day 5 at lOx magnification A. Graphical representation of KMR46 day 3 and day 5 nuclei counting for different sample groups. B. Graphical representation of KMR46 day 3 and day 5 nuclei size (pm) of different sample groups. C. Fusion of KMR46 day 3 and day 5 for different sample groups. For all graphs: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. N.s. stands for not statistically significant. (n=3) Figure 2. Differences between different samples of human FN-RMS day3 and day 5 at lOx magnification
A. Graphical representation of human FN-RMS day 3 and day 5 nuclei counting for different sample groups. B. Graphical representation of human FN-RMS day 3 and day 5 nuclei size (pm) of different sample groups. C. Fusion of human FN-RMS cells day 3 and day 5 for different sample groups. For all graphs: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. n.s. stands for not statistically significant. (n=3) Figure 3. Day 3: Changes in protein contents after treatment with MPCi and miRNA, compared to the DMSO control group
A. Changes in protein contents of MYF5 and MYOG of KMR46 samples. B. Changes in protein contents of MYF5 and MYOG of RD18 samples. For both KMR46 and RD 18, GAPDH was used as loading controls. (n=3)
Figure 4. Quantification of the protein contents based on the western blot results obtained.
A. Relative intensity of the bands of MYF5 and MYOG of KM R46. B. Relative intensity of the bands of MYF5 and MYOG of RD18. For all graphs: *p<0.05, **p<0.01, n.s. stands for not statistically significant. (n=3)
Figure 5. Nanosight batch analysis report By using the Nanoparticle tracking analysis (NTA) version 2.3. Average size/ concentration.
Figure 6. In vitro and in vivo effects of miR-449a+340 in FN-RMS. (A) Analysis on proliferation of Incucyte timelapse over the course of 144h (n=3); (B) Different effects of miRNAs on mouse FN-RMS and human mesoangioblasts (n=3); (C) EdU analysis to assess cell cycle phases on 449a340 compared to vehicle and s-PMC (n=6); (D) Rhabdosphere formation and sphere diameter after 11 days (n=3); (E)(F) In vivo effects on tumour proliferation (n=6); (G) In vitro effects on migration potential (n=3); (H) in vivo effects on metastatic potential (n=6)
Figure 7. miRNA effects on metabolism. (A) Intracellular metabolite accumulation in miRNA-treated compared to vehicle-treated FN-RMS (n=8); (B) Downregulated Gene Ontology pathways in FN-RMS patients compared to skeletal muscle; (C)Increased secretion of alanine in miRNA-treated FN-RMS (n=8); (D)Switch from glucose uptake to glucose secretion in miRNA-treated FF-RMS (n = 5).
Figure 8. MPC inhibition and oncosuppressor miRNAs similarly affect oxygen consumption rate, cell cycle and proliferation. (A) Downregulation of key genes in MPCI, MPC2 and PHGDH observed at RNAseq (n=4) (circles: vehicle: squares: mIR— 449a+340; (B) Seahorse measurement of oxygen consumption rate (n>4); (C) EdU analysis of cell cycle (n=5); (D) Nuclei counting, (E) Size, and (F)Fusion percentage in human FN-RMS. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (n = 3) Figure 9. MPCi and miRNAs similarly target in vivo migration and proliferation, and improve overall exercise capability in mice. (A) Representative image of bioluminescent signal in vivo at day 19 after the injection and (B) the photon flux from the ROIs; (C) Representative image of bioluminescent signal ex vivo from lungs and (D) the photon flux from the ROIs. N = 5. p-value: * = p<0.05; ** = p<0.01. (E) Time, (F) distance, and (G) power in mice receiving sham, vehicle-treated cells, MPCi- treated cells (MPC), and miRNA-treated cells.); (H) Western blot showing a reduced level of MYOG after MPCi or miRNA treatment, with MYF5 increasing after MPCi (n=3); * = p<0.05; ** = p<0.01; *** = p<0.001
Figure 10. Epigenetic effects of miR-449a+340 on FN-RMS. (A) RNAseq analysis shows downregulation of key genes encoding for chromatin modifiers (n=4); (B) Western Blot shows reduced levels of H3K27me3 (n=3)
2-DG: 2-deoxyglucose ; AGO: Argonaute protein; AKT: Protein kinase B; ALK: Anaplastic lymphoma kinase; Alpha-SMA: Alpha smooth muscle actin; ARMS: Alveolar rhabdomyosarcoma; ATP: Adenosine triphosphate; BCOR: BCL6 corepressor; BMPs: Bone morphogenetic proteins; BSA: Bovine serum albumin; CCND2: Cyclin- dependent kinase cyclin D2 (CCND2); CDH15/m-cadherin: Cadherin 15; CPT1A: Carnitine palmitoyltransferase ; CTNNB1 : Catenin beta 1; CXCR4: C-X-C chemokine receptor type 4; DMEM: Dulbecco's Modified Eagle Medium; DMEM-HG: High glucose Dulbecco's Modified Eagle Medium; DMSO: Dimethyl sulfoxide; ECM: Extracellular matrix; EGR1 : Early growth response protein 1; EMT: Epithelial-mesenchymal transition; ERK V2: Extracellular regulated kinases Vi ; ERMS: Embryonal rhabdomyosarcoma; ESCRT: Endosomal sorting complexes required for transport; EVs: Extracellular vesicles; EXP-5: Exportin-5 ; FAS: Fatty acid synthase ; FBS: Fetal bovine serum; FBXW7: F-box and wd repeat domain containing 7; FGFR4: Fibroblast growth factor receptor 4; FN-RMS: PAX-fusion negative rhabdomyosarcoma; FP-RMS: PAX-fusion positive rhabdomyosarcoma; GLUTs: Glucose transporters; HGFR: Hepatocyte growth factor receptor; HIF: Hypoxia inducible factors; HRAS: Harvey- ras ; HRP: Horseradish peroxidase; IGF-2: Insulin-like growth factor 2; IL-8: Interleukin-8; ILVs: Intraluminal vesicles ; KRAS: Kirsten-ras; LOH: Loss of heterozygosity; MAPK: Mitogen-activated protein kinase; MDM2: Mouse double minute 2 homolog; MF20: Myosin heavy chain antibody; MHC: Myosin heavy chain ; miRISC: miRNA-induced silencing complex; miRNAs: MicroRNAs; MPC: Mitochondrial pyruvate carrier; MPCi: Mitochondrial pyruvate carrier inhibitor; MRF4: Myogenic regulatory factor 4; MRFs: Myogenic regulation factors; MSCs: Mesenchymal stem cells ; mTOR: Mammalian target of rapamycin; MVBs: Multivesicular bodies; MVs: Microvesicles; MYF5: Myogenic factor 5; MYH3: Myosin heavy chain 3; MYOD: Myoblast determination protein; MYOG: Myogenin ; NAD+ : Nicotinamide adenine dinucleotide; NADH: Nicotinamide adenine dinucleotide hydrogen; NADPH: Nicotinamide adenine dinucleotide phosphate; ncRNAs: Non-coding RNAs; NF1 : Neurofibromatosis type 1; NRAS: Neuroblastoma-ras; P/S: Penicillin/Streptomycin; PABP1 : Poly(A)-binding protein; PBS: Phosphate-buffered saline; PI3K: Phosphatidylinositol-3-kinase; PIK3CA: Phosphatidylinositol-4,5-bisphosphate 3- kinase catalytic subunit alpha; PK: Pyruvate kinase ; pre-miRNAs: Precursor miRNAs; pri-miRNAs: Primary miRNAs; PTEN: Phosphatase and tensin homolog; RISC: RNA- induced silencing complex ; RMS: Rhabdomyosarcoma ; ROS: Reactive oxygen species; RT-qPCR: Quantitative real-time polymerase chain reaction; RTK: Receptor tyrosine kinases; SHH: Sonic hedgehog; SIX: Sineoculis homeobox homolog; SMA: Smooth muscle actin; STS: Soft tissue sarcomas; TCA: Tricarboxylic acid; TME: Tumour microenvironment; TPCs: Tumour propagating cells ; UTR: Untranslated region; WNT: Wingless and Int-1
Rhabdomyosarcoma (RMS) is a soft tissue sarcoma with characteristics of skeletal muscle lineage, having high incidence in young adults below the age of 20. There are two subtypes of RMS defined according to the presence and absence of a PAX gene rearrangement, i.e. fusion-positive RMS (FP-RMS) and fusion-negative RMS (FN-RMS), with the latter being the most common subtype of RMS.
RMS can be subdivided into six histological groups, i.e. embryonal, alveolar, spindle cell, mixed-type, pleomorphic, and RMS with ganglionic differentiation.
Pediatric RMS can be divided into two major groups, embryonal RMS (ERMS, also known as fusion-negative RMS) and alveolar RMS (ARMS, also known as fusionpositive RMS).
FN-RMS typically have a high rate of single-nucleotide alterations, and the recurrent mutation typically occurs in cancer genes such as harvey-ras (HRAS), neuroblastoma-ras (NRAS), kirsten-ras (KRAS), anaplastic lymphoma kinase (ALK), fibroblast growth factor receptor 4 (FGFR4), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), f-box and wd repeat domain containing 7 (FBXW7), neurofibromatosis type 1 (NF1), TP53, catenin beta 1 (CTNNB1) or BCL6 corepressor (BCOR). FN-RMS tumours are characterized by the loss of function TP53 mutations, and this is due to focal amplification of the mouse double minute 2 homolog (MDM2), a TP53 negative regulator. PAX3-FOXO1 has the ability to alter the transcription of genes such as FGFR4 and insulin-like growth factor 2 (IGF-2). As a result, IGF-2 overexpression occurs in FN-RMS due to loss of heterozygosity (LOH) at the llpl5.5 locus. Furthermore, in FN-RMS mutations occur in genes that affect the activation of the receptor tyrosine kinase (RTK)/RAS/phosphatidylinositol-3- kinase (PI3K) signaling axis, and somatic mutations occur in cell cycle genes, including CTNNB1, FBXW7, BCOR and p53, which commonly occur with the gain of chromosomes 2,8 and 13. The characteristics of FP-RMS are sustained activation of the RTK/RAS/PI3K signaling axis, tumours with a high copy number of the N-MYC gene (due to the PAX3-FOXO1 fusion protein regulating the oncogenic transcription of N-MYC) and a loss of imprinting at the llpl5.5 locus which leads to IGF-2 overexpression. Therefore, inhibiting the expression and/or activity of PAX3-FOXO1 by using specific inhibitors is one of the main goals of current studies for treatment of FP-RMS.
The development of an effective treatment for fusion-negative RMS (FN-RMS) is crucial, since it is the most common subtype and has a poor survival rate when it is metastatic. The combination of two oncosuppressor microRNAs (hsa-miR-449a-5p and hsa-miR-340-5p) affects the tumourigenicity of FN-RMS and the metabolism of pyruvate.
RMS cells express PAX3, PAX7 and myogenic regulatory transcription factors MRFs). PAX3 and PAX7 indicate both muscle-forming and neural crest cells. However, since RMS tissue lacks neural differentiation, expression of PAX3, PAX7 and the MRFs are then indicating the skeletal muscle origin of RMS. Depending on the oncogenes used and the cells' developmental age, FP-RMS can arise from low-passage mesenchymal stem cells (MSCs), FN-RMS from low-passage myoblasts, and undifferentiated sarcomas from both low-passage MSCs and myoblasts. Additionally, it is also possible that FP-RMS arise from developing muscle, and FN-RMS arise from muscle progenitor cells.
RMS express MRFs, such as myogenic factor 5 (MYF5) and myoblast determination protein (MyoD) [Clark et al. (1991) Br. J. Cancer 64(6), 1039-1042]. MYF5 and MyoD have important roles in regulating growth, proliferation and TPC activity in RMS. Not only is MYF5 a marker of TPCs in the zebrafish FN-RMS model, but also in vivo it can transmit tumour propagating potential to differentiated FN-RMS cells. Both MYF5 and MyoD are essential for RMS proliferation where together they regulate molecular programs that are commonly found in healthy muscle development and regeneration. The common binding site of MYF5 and MyoD is located on the promoter and enhancer regions of certain genes, such as cyclin-dependent kinase cyclin D2 (CCND2), myogenin (MYOG), and cadherin 15 (m-cadherin, CDH15'). These genes regulate cell cycle progression and muscle differentiation. This is also the reason why when MYH5 and MyoD are knocked down, the aforementioned genes are commonly downregulated. In general, MyoD and MYF5 have essential roles in muscle cell identity regulation and cell cycle regulation. Both are needed for tumour growth and may regulate stem cell self-renewal in healthy muscles.
Mitochondrial pyruvate carrier and cancer metabolism
Looking at the physiology of muscle, fast-twitch muscle fibres generate energy with a high anaerobic glycolytic rate, whereas slow-twitch fibres consume lactate to regenerate pyruvate as supply for the TCA cycle. In tumours, when glucose is limited, the most oxygenated cancer cells consume lactate for their metabolism. The spared glucose can then diffuse to the most hypoxic cancer cells to support their survival. Mitochondrial pyruvate carrier (MPC) consists of two parts, MPC1 and MPC2, which are gatekeepers for pyruvate transportation and oxidation in mitochondria. In cancer, the blockade of MPC activity promotes cytosolic accumulation of pyruvate which stops the intracellular production of pyruvate through lactate conversion and prevents the uptake and consumption of extracellular lactate by cancer cells. Therefore, when lactate was the only available nutrient, the blockage of MPC activity prevented cancer cell growth. Also, the MPC inhibition could force cells to go from partially aerobic glucose metabolism to strict anaerobic glycolysis which could also cause cytotoxic effects. Finally, when blocking lactate and glucose from entering the TCA cycle as fuel, the inhibition of MPC activity also massively decreases the oxygen consumption and thus sensitizes tumours to radiotherapy via local reoxygenation. However, the blockade of MPC activity leads to the inhibition of lactate uptake, but also alters glucose oxidation.
The depletion or blocking of MPC activity could lead to aerobic glycolysis and induce growth of tumour cells. A lower expression of MPC proteins has been shown to lead to poor survival rates in patients with different types of cancers, such as colon cancer, prostate cancer, and oesophageal cancer.
Depending on the stage of tumour development and the tumour microenvironment, cancer cells undergo metabolic status changes where cells adapt to prevent cell death and to sustain continuous cell division. One important characteristic of cancer metabolism is that tumour cells tend to metabolize glucose anaerobically, even in aerobic conditions. Compare to the aerobic glycolysis, this type of metabolism leads to increase in glucose uptake and production of lactate via fermentation of glucose.. This type of molecular adaptation is called the Warburg effect where glucose-derived carbon skeletons are used for macromolecule biosynthesis instead of oxidation via the TCA cycle in the mitochondria. In the pentose phosphate pathway, cancer cells alternatively use the glucose backbone to produce ribose and nicotinamide adenine dinucleotide phosphate (NADPH) for nucleotide synthesis, lipid biosynthesis and the maintenance of the cells' redox state. Increases in the Warburg effect led to decreased levels of o-ketoacid intermediates entering the TCA cycle which, as a result, increases the demand of glutamine for cancer cells. Glutamine is a non- essential amino acid precursor, a substrate for gluconeogenesis, and also an alternative energy source for rapidly dividing cells. Through glutaminolysis, glutamine turns to glutamate which becomes oxaloacetate that can enter the TCA cycle and act as a partial glucose replacement for cancer cells. In most human cancers, high expression levels of fatty acid synthase (FAS) are commonly found. FAS is an important metabolic enzyme which can catalyse the synthesis of long-chain saturated fatty acids to support the demands of membrane biogenesis.
Metabolic changes in RMS
In cancer cells, an upregulated embryonic M2 form of pyruvate kinase (PK), which has an insufficient catalytic ability to produce pyruvate, has oftentimes been found. Therefore, as an alternative substrate, oxidation of glutamine/glutamate occurs to produce pyruvate and then via lactate production to regenerate nicotinamide adenine dinucleotide (NAD+).
By using [U-13C]- glucose as a marker and via isotopomer-based metabolomic analysis, major differences in central energy and anabolic metabolism have been found between the primary myocyte and transformed Rh30 cell lines (human RMS cells). Compared to the myocytes, in the Rh30 cells, glycolysis, the Krebs' cycle, the pentose phosphate pathway, and nucleotide biosynthesis were found to be enhanced in order to support the accelerated growth of tumours. In Rh30 cells, the balance between energy production and the biosynthetic precursor production may be regulated by an activation of anaplerotic carboxylation and the malate/aspartate shuttle which promotes nicotinamide adenine dinucleotide hydrogen (NADH) hydride to migrate from the cytoplasm to the mitochondrion for oxidative phosphorylation.
PAX3-FOXO1 coordinates the expression of several PAX3 downstream genes, such as hepatocyte growth factor receptor (HGFR or c-MET), FGFR4, IGF-2 and C-X-C chemokine receptor type 4 CXCR4), which are key genes involved in the increase in tumour aggressiveness and metastasis recurrence of FP-RMS. When looking at the metabolic processes, PAX3-F0X01 regulates the transcription of the GLUT4 gene which leads to an increase of glucose consumption in FP-RMS cells. Therefore, using 2-deoxyglucose (2-DG) as a glycolytic inhibitor for cancer treatment has caused death in several FP-RMS cell lines, which indicates that an increase in glucose consumption due to PAX3-FOXO1 can be rate-limiting for tumour growth. Furthermore, PAX3-FOXO1 also causes the downregulation of the phosphate and tensin homolog deleted on chromosome 10 PTEN) gene [101], thus resulting in the activity alteration of the PI3K/(protein kinase B)AKT/mammalian target of the rapamycin (mTOR) pathway). Together with the activity of AKT, transcription of the carnitine palmitoyltransferase gene CPT1 A) forms acyl carnitines which facilitate lipid degradation. This could be the source of energy for FP-RMS cells for migration and metastasis.
Both FN-RMS and FP-RMS are related to the activation of RTK-dependent pathways, which include the PI3K/AKT and RAS pathways. The PI3K/AKT pathway triggers the consumption of glucose, amino acids and other nutrients. With the activation of mTOR, glycolysis and lactate production, lipogenesis and other protein synthesis are promoted.
With the outcome of the PI3K/AKT pathway and mitogens, activation of Myc transcription factors initiates the RAS pathway. The RAS pathway controls the expression of different genes and microRNAs and thus leads to cell division, formation of tumours and changes in metabolism, such as the Warburg effect, lipogenesis and glutamine anapleurosis. Furthermore, the AKT/mTOR pathway activates the Hypoxia inducible factors (HIF). HIF is responsible for helping solid tumours survive in a low oxygen environment.
Through the observation of different FN-RMS and FP-RMS cell lines under hypoxia, the activation of the PI3K/AKT and extracellular regulated kinases 1/2 (ERK 1/2) pathways leads to the increase in formation of the proangiogenic interleukin-8 (IL- 8), which then increases the cell survival rate. When the IL-8 molecule reacts with two 7-transmembrane G-protein-coupled receptors, i.e. CXCR1 and CXCR2, it takes the role of a mitogenic autocrine growth factor for some human cancers.
Under normal circumstances regulators such as p53, AKT, MYC and HIF proteins regulate the glucose metabolism in healthy tissues. In particular, p53 has a role in inhibiting the expression of glucose transporters (GLUTs), for example GLUT1 and GLUT3/GLUT4, and it reduces glucose consumption in the glycolytic pathway. p53 also regulates the expression of PTEN which negatively regulates glycolysis and the PI3K/AKT/mTOR and HIF pathways. Therefore, for cells with no p53 activity, typically high sugar transport across the cell membrane and high glucose consumption are found. The chances to have a loss of p53 activity are higher in FN-RMS than in FP- RMS, with an incidence rate of 5-19%. As an example, in the human RD cell line, glucose consumption is higher since the expression of the GLUT1 and GLUT4 increase due to the loss of p53 activity. Additionally, increasing the expression of GLUT1 transporter increases glucose consumption and glycolytic use which eventually is the reason for high cell proliferation and invasiveness.
Activation of the RAS pathway is closely related to FN-RMS aggressiveness. Typically, RAS-positive FN-RMS cells are related to increased oxidative stress. FN-RMS tumours are found to have a high rate of G to T transversions due to oxidative damage. Furthermore, FN-RMS promote methylation of certain genes which regulate metabolism, mitochondrial function and oxidative stress. According to the studies on lung tumours, activation of RAS pathways will increase the production of mitochondrial reactive oxygen species (ROS) levels and thus lead to an increase in cancer cell growth through the activation of mitogen activated protein kinase (MAPK). As a result, FN-RMS tumours could benefit from increased ROS levels, but at the same time, increasing accumulation of ROS-induced gene mutations could lead to cell death as well. Therefore, oxidative stress inducer, actinomycin-D could be the key to control tumour growth.
MicroR As
MicroRNAs (miRNAs) belong to the class of non-coding RNAs (ncRNAs), with an average size of 22 nucleotides and having roles in regulating gene expression. Most miRNAs are first transcribed from DNA into primary miRNAs (pri-miRNAs), then inside the nucleus pri-miRNA is further processed into a precursor miRNA (pre-miRNA) by a class 2 RNase III enzyme called Drosha. After this, exportin-5 (EXP-5) helps with the transportation of pre-miRNA to the cytoplasm via nuclear pore complexes, a large proteinaceous channel located in the nuclear membrane. Inside the cytoplasm, pre- miRNAs are processed by Dicer to become mature miRNAs. Dicer, an RNase III type protein, is a very specific type of enzyme that measures and cleaves 22 nucleotides of the miRNA strand, and it appears in almost all eukaryotic organisms. There are multiple types of Dicer of which Dicer-1 is responsible for miRNA maturation and Dicer-2 is responsible for siRNA maturation. Finally, the mature miRNAs are loaded onto the Argonaute (AGO) protein, which forms the effector RNA-induced silencing complex (RISC). In most cases, one strand of the miRNA is degraded while the other strand remains attached to Ago as mature miRNA. This selection of strands is based on the thermodynamic stability of the ends of the duplex. After miRNAs are loaded, RISC is guided by the miRNA to the target mRNA where silencing occurs via degradation or translational repression.
To induce mRNA degradation and translational repression, miRNAs interact with the target mRNAs via the 3' untranslated region (3' UTR), or in some cases, via other sites such as the 5' UTR, a coding sequence, and gene promoters. Depending on the situation, miRNAs can activate translation or regulate transcription as well. However, interaction of miRNAs with target genes can be affected by many factors, for example, the subcellular location of miRNAs, the abundancy of miRNAs and mRNAs and the affinity of their interactions. miRNAs secreted into extracellular fluids can be transported to target cells by vesicles. Extracellular miRNAs act as chemical messengers which can mediate cell-cell communications.
According to current studies, it seems that the eIF4F complex is related to the initiation of translation. This complex consists of subunits, such as eIF4A, eIF4E, and eIF4G. First, the eIF4E subunit recognizes the mRNA 5' terminal cap and starts the initiation process. Then, to enable the preinitiation complex, another initiation factor, eIF3, reacts with eIF4G and helps with the 40S ribosomal subunit assembly at the 5'end of the mRNA. The elongation process is then initiated by the 60S ribosomal subunit and 40S preinitiation complex binding at the AUG codon of the mRNA. Additionally, eIF3 and eIF4G also react with the poly(A)-binding protein (PABP1). Finally, as a result, mRNA molecules become circular and thus the translational efficiency is improved. However, the mechanism of translational repression is still not clear, more specifically, at which translational level repression occurs is still unknown. According to previous research, miRNA-induced silencing complex (miRISC) may repress the elongation process, and it could promote early ribosome dissociation from mRNAs. There are several possible mechanisms where miRISC could represses the initiation mechanism. First, miRISCs are competitive with eIF4E for the binding to the mRNA 5' cap structure which would lead to an unsuccessful translation initiation process. Second, miRISC prevents the mRNA from turning into a circular shape which causes translational inhibition. Third, miRISC can inhibit the 60S ribosomal subunit from joining with the 40S subunit, leading to translational repression. Lastly, miRNA or miRISC can have an effect on translational repression due to the increase in target mRNAs in the processing bodies.
The degradation of target mRNAs is induced by the AGO protein slicer activity. miRNAs also affect this process. Some other mechanisms are also involved, such as deadenylation, decapping and exonucleolytic digestion of mRNA. However, the number, type, and position of mismatches in miRNA and mRNA duplexes are essential for the selection of the degradation or translational repression mechanisms. miRNAs and myogenesis
During the process of myogenesis, several genes regulate the different stages of cell maturation which eventually results in muscle tissue formation. Muscle development is regulated by the MRFs: MYF5, MyoD, MYOG and MRF4 which promote the formation of muscle fibres. miRNAs in this case have an important role in the development of skeletal muscle tissues [Buckingham (2001) Curr Opin Genet Dev 11, 440-448]. For example, some typically upregulated miRNAs, such as miR-133a-l, miR-133a-2, miR-133b and miR-206, have an important role in myogenic differentiation [Chen et al (2006) Nat Genet 38, 228-233; Kim et al. (2006) J Cell Biol 174, 677-687]. miRNAs in muscle tissue can be subdivided into two types, muscle-specific myo- miRNAs and non-myomiRNAs which are found in both skeletal and cardiac muscles [Gasparini et al. (2019) Int J Mol Sci 20, 5818].
In the differentiated skeletal muscle, muscle-specific myo-miRNAs can be either uniformly expressed over muscles (i.e., miR-1 and miR-133a), or can be highly expressed in slow-twitch/type I muscles (i.e., miR-206, miR-208b and miR-499). Myo-miRNAs correspond to only 3% of expressed miRNAs in quiescent satellite cells, but in differentiated cells myo-miRNAs correspond to 74% of expressed miRNAs. In general, during the myogenic development, regulation is needed to activate specific miRNAs at different stages of myogenesis to achieve optimal balance between proliferation and myoblast differentiation. miRNAs as a biomarker and the role in RMS development miRNAs can regulate post-transcription of different phases of muscle-specific processes, which shows their ability as biomarkers in the development of RMS [Bersani et al. (2016) cited above]. The myo-miRNAs are the key to skeletal muscle tissue differentiation, where their downregulation is related to the dedifferentiation of the phenotype in RMS [Novak et al (2013) Pediatr Blood Cancer 60, 1739-1746]. The overexpression of miR-1 in RD cell lines regulates the gene expression of muscle tissue and cell cycle arrest, whereas miR-133a decreases the expression of muscle markers. For RMS, miR-1, miR-22 and miR-206 are downregulated while miR-29 is deregulated in a few FP-RMS [Bersani et al. (2016) Cancer Res 76, 6095-6106]) .
Since miRNAs have important effects on the development and progression of RMS, these molecules could be interesting as therapeutic targets in pediatric tumours. For example, overexpression of miR-378 can cause changes in apoptosis, migration, and viability. miR-183 has an oncogenic role and it targets two tumour suppressor genes, specifically early growth response protein 1 EGR1) and PTEN. Extracellular vesicles - Exosomes
Extracellular vesicles (EVs) are mediators of cell-cell communication and play an important role in cancer metastasis. Therefore, their use as biomarkers for clinical purposes is being explored extensively . EVs are lipid bilayer vesicles with a diameter size of 40-1000 nm which are produced and released by cells or detached from the plasma membrane. EVs can be divided into subtypes based on their biogenesis, release pathway, size, content, and function. The three main subtypes of EVs are microvesicles (MVs), exosomes, and apoptotic bodies.
Exosomes or intraluminal vesicles (ILVs) have a single outer membrane and are secreted by all types of cells. Exosomes can be found in many fluids such as plasma, urine, saliva, or tears. Exosomes have a diameter size of 30-150 nm and are formed via an endosomal route. Early endosomes formed from inward budding of the cells' plasma membrane, later mature into multivesicular bodies (MVBs) which are involved in protein sorting, recycling, storage, transport, and release. Then, MVBs are either degraded by the lysosomes or fuse with the cells' plasma membrane to secrete exosomes and other contents into the extracellular space. The fate of a MVB depends on the level of cholesterol it contains. Vesicles rich in cholesterol are secreted while vesicles with a low cholesterol level are degraded by the lysosomes. The formation of MVBs is promoted by growth factors. The cell adapts its production of exosomes based on its needs.
When looking at the biogenesis of exosomes, exosomal formation and MVB transportation are regulated by endosomal sorting complexes required for transport (ESCRT) proteins and with accessory proteins. These are commonly considered as "exosomal marker proteins". These "marker proteins" can be found in all exosomes, independent from cell type. The transmembrane proteins CD63, CD9 and CD81, belong to the tetraspanin family, which are also often found in exosomes.
The biological purpose of exosomes includes cell-cell communication, cell maintenance and tumour progression. Furthermore, when exosomes act as antigen- presenting vesicles, immune responses can be stimulated. Exosomes in the nervous system can promote myelin formation, neurite growth, and neuronal survival, which in the end also help with tissue repair and regeneration.
Until now, a lot of research has been done to investigate the ability of exosomes as carriers of biomarkers for different diseases. For example, specific exosomal markers for pancreatic cancer and lung cancer have been found. Since exosomes can be found in bodily fluids, it is optimal to use exosomes as carriers of biomarkers which can lead to non-invasive diagnostics and the monitoring of a patients' response after treatment. Exosomes are also suitable for immunological applications since they have a relatively long circulating half-life, are tolerated by the human body, are able to penetrate cellular membranes, and have the potential to target specific cell types. One of the important properties of exosomes is its inherent property, which can act inherently as antigen presenting vesicles, and makes exosomes ideal to use as a drug delivery system. Nucleic acids and proteins can be introduced into exosomes which can be further modified to target specific cells.
A combination of oncosuppressor microRNAs (miR-449a+miR-340) was identified, which showed downregulation of MPCI and MPC2 upon treatment of FN-RMS models with these miRNAs.
In addition the effects of MPC inhibition on human and murine FN-RMS were tested and compared to oncosuppressor miRNAs by means of immunofluorescence staining, RT-qPCR and western blot. Furthermore, the isolation and analysis of EVs was performed which allows the use of EVs as carriers of oncosuppressor miRNAs in FN- RMS models in vivo.
RMS is a type of STS, and the most common type in children. RMS tumours are histologically similar to normal foetal skeletal muscle. As mentioned, there are two major subtypes of RMS: FP-RMS and FN-RMS. Looking at the morphology, FP-RMS typically are small round densely packed cells, whereas FN-RMS typically consist of spindle-shaped cells and are rich in stroma. Herein, the effects of targeting energy metabolism via the mitochondrial pyruvate carrier inhibitor (MPCi) are investigated as well as specific genes via miRNAs in FN-RMS tumour cells using RT-qPCR, western blot and immunofluorescence microscopy techniques.
For both murine KMR46 and human RD18 FN-RMS cells, a decrease in the confluency was found in the MPCi- and miRNA-treated groups. Furthermore, the MPCi-treated group showed elongation of cells and intention to form multinucleated-like structures, whereas in the miRNA-treated group, there were formations of gigantic mono-, bi-, tri- or multinucleated cells instead more visible in murine FN-RMS. This could be due to the senescence effects, which led to a lower cell confluency and formation of larger cells. From these results, a possible preliminary conclusion could be that the MPC inhibition has effects of inhibiting tumour growth or proliferation, by altering cancer metabolism. In tumours, when glucose is limited, the most oxygenated cancer cells consume lactate instead for their metabolism. However, MPC inhibition leads to a blockade in the uptake and consumption of extracellular lactate, which could also force cells to go from a partially aerobic glucose metabolism to a strict anaerobic glycolysis, causing cytotoxic effects. Therefore, this ca, explain why there was a decrease in cell number and less proliferation after treatment with MPCi compared to the DMSO control group.
Literature shows that the decrease in MPC activity can lead to aerobic glycolysis and induce the growth of tumour cells, metastasis, and poor survival of patients with different types of cancers, such as colon cancer, prostate cancer, and esophageal cancer. Thus, an planation for MPCi having possible anti-cancer effects instead of pro-cancer effects is that the role of MPC is highly dependent on the tissue type and metabolic inclinations of cancers. Since colon, prostate, and esophageal cancers arise from epithelial cells, whereas RMS can arise from mesenchymal stem cells or muscle progenitor cells, there might be an effect on the response of MPC inhibition. In general, cancers that use mitochondrial pyruvate for tumour growth and glutamine for glutathione production are typically negatively affected by MPCi.
RMS tumours express myogenic regulatory transcription factors, such as MYF5 and MyoD, which have important roles in regulating growth, proliferation and tumour propagating cell activity in RMS. Furthermore, the binding sites of MYF5 and MyoD are located on the promoter and enhancer regions of some genes, such as MYOG, which regulate cell cycle and muscle differentiation. Indeed, the effect of MPCi treatments on both KMR46 and human FN-RMS samples affected MYF5/ Myf5, MyoD, and MYOG/ Myog expressions as indicated by the RT-qPCR analysis. In the KMR46 samples, after MPCi treatment, Myf5 was upregulated whereas MyoD and Myog were downregulated. For the human FN-RMS samples, MYF5 was undetermined, MyoD was upregulated, and MYOG was again downregulated. These results indicate that the downregulation of either MYF5/Myf5 or MyoD could lead to the downregulation of MYOG/Myog. According to literature, MYF5 and MyoD share the same binding site but due to functional divergence, each established a separate muscle lineage. Therefore, regulation of either MYF5 or MyoD could lead to changes in skeletal muscle differentiation. For example, regulation of MRFs due to MPC inhibition may promote cells to differentiate into skeletal muscle-like cells whereas the miRNA-treated cells are more likely to differentiate into smooth muscle-like cells.
There are two parallel paths where cells expressed either MYF5 or MYH3 and terminated with expressing either MYOG or MYH3. Therefore, an explanation for the effect of MPCi treatment can be that it saves the cells from being trapped in the MYF5 path, by regulating the expression of MYF5 and MyoD. Thus, the cells either go down the same path or by another path, which eventually leads to a more successful myogenesis.
The regulation of MYF5 and MYOG translation was further investigated via western blot analysis. Looking at the protein content, in most cases the results showed changes of protein content between treated and untreated samples. However, when looking at the statistical analyses of the results, in most cases the content changes were not statistically significant.
The tumour microenvironment (TME) consists, besides tumour cells, of fibroblasts, endothelial cells, immune cells, and non-cellular components (including exosomes and cytokines) which form an internal environment where tumour cells survive. Thus, the TME plays an important role in all stages of tumourigenesis. miRNAs can be carried by exosomes, which have an important role in intercellular transport and signalling. The heterogeneity of the TME is mainly due to miRNAs' activation of cancer-associated fibroblasts which also reshape the extracellular matrix (ECM) and thus promote the spreading of tumour cells. Therefore, using extracellular vesicles (EVs) or exosomes as carriers for oncosuppressor miRNAs could be an interesting way to alter the proliferation and metabolism of tumour cells.
EXAMPLES
Example 1 Materials and methods
Cell Culture
Human RD18 and murine KMR46 FN-RMS cell lines were cultured in high glucose Dulbecco's Modified Eagles Medium (DMEM-HG) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (P/S). Cells were maintained under standard incubator conditions humidified atmosphere (95% air, 5% CO2, 37°C) and passaged twice weekly with 0.25% Trypsin-EDTA.
Incucyte timelapse imaging
Cells were trypsinized and plated at 3000 cells per well in a 96-well plate (Corning). miRNA treatment and PI staining was added to each well, photos of each well were taken every 2 h for 144 h using Incucyte Live Cell Imager (Essen Bioscience) and cell confluence was measured by Incucyte Software (Essen Bioscience). Co-cultures
For co-culture experiments, cells were seeded at 1 :20 KMR46/human MABs ratio on collagen-coated vessels in IMDM 15% FBS medium, 1% L-glutamine, 1% non- essential amino acids, 1% sodium pyruvate, 1% ITS, 1 : 10,000 basic FGF, 0.2% betamercaptoethanol. After 24 h, medium was changed and OptiMEM with miRNA- lipofectamine or lipofectamine was added to the wells. A total of 24 h after miRNA addition, cells were differentiated in DMEM 2% horse serum medium for 96-120 h in 5% 02/5% CO2 at 37°C.
Reporter Cell Line
Cells were transfected with lentiviral particles expressing EFla-eGFP-P2A-fLuc and selected with 1 pg/mL puromycin for 2 weeks. GFP was used as a standard gene expression tracer in vitro. Firefly luciferase (fLuc) was used as an optical reporter gene, upon coelenterazine administration, to detect the cell line engraftment in vivo via IVIS bioluminescence imaging (BLI).
MiRNA/MPCi treatment
Before transfection, cells were seeded at 12-15.000 or 50.000 cell number in 24- wells or 6-wells, respectively. One day after plating, cells were transfected using 3 pl/mL Lipofectamine 2000 and 1.6 pl/mL of hsa-miR-449a-5p/-340-5p, or hsa-miR- 181a-5p/-212-3p (all MISSION® microRNA Mimics, Sigma-Aldrich), at 10 pM concentration resuspended in OptiMEM-medium. For MPCi treatment, cells were treated with 8 pl (final concentration is 40 pM) of Mitochondrial pyruvate carrier inhibitor (UK-5099, Sigma-Aldrich) in 2 ml of Opti-MEM™ I Reduced Serum Medium. For the control group, cells were treated with 8 pl of Dimethyl sulfoxide (DMSO) (Sigma-Aldrich) in 2 ml of Opti-MEM™ in Reduced Serum Medium. After 60-65 h of transfection, cells were trypsinized, counted and either frozen or employed for subsequent experiments.
Immunofluorescence
Following [Ronzoni et al. (2021) Int J Mol Sci. 22(4), 1939], fixed cells were permeabilized with 1% BSA, 0.2% TritonX-100 in PBS for 5 min and then blocked in 10% donkey serum for 1 h. After 1.5 h of incubation with primary antibodies, diluted in 1% donkey serum, samples were washed three times with PBS, incubated with anti-mouse or anti-rabbit secondary antibody (1: 1000) conjugated with 488 or 594 AlexaFluor fluorochromes (Invitrogen Milan, Italy) and nuclei were counterstained with Hoechst 33258 at 1 pg/ml (Sigma, Italy). Here follows the list of primary antibodies and relative dilutions: mouse anti-MyHC (DSHB #MF20), 1 :3; rabbit antilamin A/C (Epitomics #2966-1), 1:600; mouse anti-Myogenin (Invitrogen #AB_10977211), 1: 100; rabbit anti-MyoD (Thermo Fisher Scientific #MA5-12902), 1: 100. Imaging was performed at Eclipse Ti microscope (Nikon) by means of Image- Pro Plus 6.0 software (Nikon).
Rhabdospheres
20,000 pretreated cells were seeded into 6-well Ultra-Low Attachment plates (Corning, USA) in serum-free DMEM/F12 (Invitrogen, USA), supplemented with B27 (lx, Invitrogen, USA), 20 ng/ml EGF (Peprotech, USA), 20 ng/ml bFGF (Invitrogen), and 1% Penicillin/Streptomycin (Sigma, USA). Pictures were taken every day for 11 days.
Oxygen Consumption Rate
Seahorse XFp Extracellular Flux Analyzer was employed to measure oxygen consumption rates (OCR) using the Mito Stress Test Kit following the manufacturer's protocol. Briefly, pretreated 40,000 cells/well were plated Seahorse XFp cartridge for 4 hours in growth medium. Following attachment to the well, medium was changed with XF media pH 7.4 supplemented with 2mM L-Glutamine, 10 mM glucose and 1 mM sodium pyruvate. Basal OCR was measured for 4 cycles, then 8 pM oligomycin was injected to inhibit ATP-linked respiration, followed by 4.5 pM FCCP to measure maximal respiration, and finally 10 pM rotenone/antimycin A was injected to completely inhibit all mitochondrial respiration. After each experiment, protein concentration was measured and wells were normalized using the Wave software.
EdU assay
EdU staining was performed using Click-iT® EdU Alexa Fluor® 647 Flow Cytometry Assay according to manufacturer's advice. Briefly, after EdU incubation of l-2h, cell pellets were fixed with 4% PFA, permeabilized with 0.5% Triton X-100 in PBS for 20 min, and subsequently treated with Click-iT reaction cocktail at room temperature for 10 minutes. Subsequently, cells were washed and incubated in propidium iodide (PI) staining buffer (20 pg ml-1 PI, 0.2 mg ml-1 RNase A, PBS) for 30 min at room temperature. Cell cycle distribution was analysed by flow cytometry (BD FACSCanto HTS) and FlowJo software.
Transwell Migration Assay
To examine the migration potential following miR pre-treatment, following the previous titration [Camps et al. (2020) Cell Rep. 31(5), 107597]. 50,000 cells were seeded in the top chambers of a transwell in serum-free medium (#3422, Corning). As chemoattractant, the lower compartment contained medium supplemented with 10% FBS. Cells were incubated for 24 h and cells that did not migrate through the pores were removed by a cotton swab. Filters were fixed in 4% formaldehyde solution and stained with 0.1% crystal violet. Five pictures per filter were taken and cell number was counted using ImageJ. Drug screening
Cells were seeded in 96-well, and 24 hours later tazemetostat (EPZ-6438, HY-13803, MedChemExpress), doxorubicin (D1515-10MG, Sigma Aldrich) or topotecan (HY- 13768, MedChemExpress) were added at various concentrations and viability was measured at 24h, 48h, 72 h and 96h using the XTT assay (X6493, Invitrogen) following the manufacturer's instructions. The vehicle (DMSO; D8418, Sigma Aldrich) was used as a negative control. Dose-response curves and IC50 values were calculated using Graphpad Prism (Version 9.1.0).
RNA Isolation and RNAseq
Total RNA of each sample was extracted using PureLink RNA Mini Kit (Ambion) and DNase I treatment was performed using the DNA-free kit (Ambion). Reverse transcription was performed using the Superscript III First-Strand Synthesis SuperMix (Invitrogen). RNA-sequencing libraries were constructed with the Lexogen library. Samples were indexed with unique adapters and pooled for single read (50 bp) sequencing in Illumina HiSeq2000. RNA-seq reads were aligned with TopHat v2.0.2 to the mouse genome mmlO. Transcripts were assessed and quantities were determined by Cufflinks38. Differential expression levels were assessed using DESeq2. Gene Ontology Biological Process (GO: BP) pathways were identified using g:Profiler [Raudvere et al. (2019) Nucleic Acids Res 47(W1):W191-W198]. Data has been deposited in GEO under accession code GSE175816.
ATACseq
Sequencing reads were trimmed and aligned to assembly GRCh38 using bwa mem. Duplicate reads and reads mapping to mitochondrial sequences were subsequently removed. Chromatin accessibility peaks were called using MACS2 and annotated using HOMER and/or ChipSeeker. Differential accessibility analysis was performed using the R/Bioconductor package DiffBind.
Bioluminescence Imaging for in vivo Tumour Engraftment and Growth
C57/BI6 mice were injected in the femoral artery with either 1 x 105 KMR46 Fluc+ cells (untreated group) or with 1 x 105 KMR46 Fluc+ cells pretreated for 3 days with s-PMC (treated group). For the injection, cells were suspended in 50 pl saline water. Afterward, the mice were monitored through BLI every other day starting from day 7 after tumour injection for 10 days. For in vivo BLI scans, mice were placed in the flow chamber of IVIS® Spectrum. Subsequently, 126 mg/kg of D-luciferin was injected subcutaneousl. Hence, consecutive frames were acquired until the maximum signal intensity was reached. Pulse/sec intensities were calculated by comparing the same ROI for all the animals, after subtracting the background signal coming from not injected mice.
Treadmill Exhaustion Test
A group of five C57/BI6 mice injected in the femoral artery with 1 x 105 KMR46 Fluc+ cells (untreated group) and five C57/BI6 mice injected with 1 x 105 KMR.46 Fluc+ cells pretreated for 3 days with s-PMC (treated group) underwent functional tests by the treadmill exhaustion test. The test was performed at day 7, 9, 11, and 13 after the beginning of the experiment (day 0). The electric shock frequency and intensity were pulses of 200 ms/pulse of electric current with 2 pulse/s repetition rate (3 Hz) and intensity (1.22 mA). The mice were introduced to the treadmill belt and an adaptation time of 5 min was given before the recordings (motor speed set to zero, for 5 min). A training time of 2 min at 4 m/min was set. Later on, the motor speed was set to 7 m/min, with a 1 m/min increase and a constant uphill inclination of 20°, until exhaustion and >10 s stop. The mice were weighted right after every run. Speed (m/min), distance (m), and time (min and s) were registered and used for calculating the work of each run in J. The formula here applied was: Work (J) = body mass (kg) x gravity (9.81 m = s2) x vertical speed (m/s x angle) x time (s).
Statistical Analysis
Sample size for in vitro/in vivo experiments was calculated by means of Sample Size Calculator (parameters: power, 0.80; alpha, 0.05). When applicable, sample size analysis was based on average values obtained from preliminary optimization/validation trials. Depending on the experiment, two-tailed unpaired Student's T-tests analysis or two-way ANOVA test was performed. All statistical analyses were conducted using Prism v9.1.0 (GraphPad).
Cell lines
Human FN-RMS (RD and RD18) and murine FN-RMS (KMR46) cell lines were used. RD and RD18 are different cell lines but both are human FN-RMS with characteristics of expressing MYOG, p21 and Desmin. When looking at the morphological behaviour of RD and RD18 cells, the cells have a tendency to fuse and form myotubes and have a relatively low metastatic potential compared to other cell lines. KMR46 is a cell line derived from the mouse model MyoD-cre x KRASG12D x TP53F/-). When analyzing the bulk RNA sequencing results, the KMR46 cell line has similar characteristics to FN- RMS patients, which indicates it is a suitable model to use for this research.
Cell culture and transfection
Human RD18/RD and murine KMR46 FN-RMS cell lines were cultured in high glucose Dulbecco's Modified Eagle Medium (DMEM-HG) (Gibco™, Thermo Scientific™, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (P/S) (Gibco™). The medium was filtered with 0.22|jm Stericup® Filter Units (Merck Millipore, Burlington, MA, USA). Cells were maintained under standard incubator conditions, a humidified atmosphere (95% air, 5% CO2, 37°C) in T-75 flasks (Thermo Scientific Nunc™ EasyFlask 75 cm2 Nucleon™ Delta Surface, Thermo Scientific™) and passaged twice weekly with 0.25% Trypsin-EDTA (Gibco™). Before transfection, cells were seeded at 12-15.000 or 50.000 cells in 24- wells or 6-wells (Costar™, Corning Incorporated, Corning, NY, USA), respectively. One day after plating, cells were transfected using 3 pl of lipofectamine 2000 (Invitrogen™, Thermo Scientific™) and 1.6 pl/mL of hsa-miR-449a-5p and hsa-miR- 340-5p mimics (Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 10 pM, resuspended in Opti-MEM™ I Reduced Serum Medium (Gibco™). For MPCi treatment, cells were treated with 8 pl (final concentration is 40 pM) of Mitochondrial pyruvate carrier inhibitor (MPCi - UK5099) (Sigma-Aldrich) in 2 ml of Opti-MEM™ I Reduced Serum Medium. For the control group, cells were treated with 8 pl of Dimethyl sulfoxide (DMSO) (Sigma-Aldrich) in 2 ml of Opti-MEM™ I Reduced Serum Medium. After 60-65 hours of transfection, cells were trypsinized, counted and either frozen or employed for subsequent experiments.
Immunofluorescence staining
Fixing of the cells
Cells from day 0, day 3 and day 5 of KMR46 and RD18 plates, as mentioned previously, were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 10 minutes at room temperature and then washed three times with phosphate-buffered saline (PBS) (Gibco™). For 6-well plates, each well was maintained with 2ml of PBS. Then the plates were sealed with parafilm and stored at 4°C.
Cells were permeabilized with PBS containing 0.2% Triton X-100 (Sigma-Aldrich), for 30 minutes at room temperature. After removal of the triton solution, cells were blocked with a 1 : 10 dilution of donkey serum in PBS containing 1% bovine serum albumin (BSA) (Sigma-Aldrich), for one hour at room temperature.
Cells were then incubated with primary antibodies: 1:20 MF20 (Myosin heavy chain) (mouse) and 1:200 Ki-67 (rabbit) (Abeam, Cambridge, United Kingdom, ab833) overnight at 4°C. Then on the second day, primary antibodies were removed and the wells were washed three times with PBS and incubated with secondary antibodies: 1 :500 Alexa-488-conjugated donkey anti-rabbit (Thermo Fisher Scientific Invitrogen™, AB_2556546) and 1 :500 Alexa-594-conjugated donkey anti-mouse (Thermo Fisher Scientific Invitrogen™, AB_2556543), for one hour at room temperature, in the dark. Other wells were incubated with Cy3-conjugated 1 :200 alpha-SMA (smooth muscle actin) (rabbit) (Sigma-Aldrich, C6198) and 1:500 phalloidin (Sigma-Aldrich, P1951) for one hour at room temperature, in the dark. After removal of the secondary antibodies, wells were washed three times with PBS. To stain the cell nuclei, cells were incubated with 1: 1000 Hoechst dye (Thermo Scientific™, #62249), for five minutes at room temperature, in the dark. Finally, after removal of the dye, wells were washed three times with PBS, and after the final wash, 2 ml of PBS were added to each well for preservation purposes.
For the visualization of cells, a Nikon Eclipse Ti-S microscope (Nikon, Minato, Tokyo, Japan) was used and the collected images were further processed with the Image J software (National Institutes of Health (NIH, Bethesda, MD, USA).
Quantitative real-time polymerase chain reaction (RT-qPCR)
RNA isolation and cDNA synthesis
Cells were collected in 1.5 ml Eppendorf tubes, lysed in lysis buffer (PureLink® RNA mini kit, Ambion® by life technologies™, Thermo Fisher Scientific Invitrogen™), supplied with 1% 2-mercaptoethanol (Sigma-Aldrich). RNA was extracted from cells by using the PureLink® RNA mini kit (Ambion® by life technologies™, Thermo Fisher Scientific Invitrogen™), and by following the manufacturers' instructions.
To ensure a good-quality RT-qPCR, a DNase I treatment was done by using the Ambion® DNA-free™ kit (Ambion® by life technologies™, Thermo Fisher Scientific Invitrogen™). Where 0.1 volumes (vols.) of lOx DNase I buffer and 1 pl of DNase I were added to the RNA, and incubated at 37°C for 20-30 minutes. 0.1 vols. of DNase inactivation Reagent were added afterwards, and samples were incubated for 5 minutes at room temperature, with mixing every minute. After centrifugation, the RNA/supernatant was collected.
The RNA collected after the DNase I treatment was then used for cDNA synthesis, by using a cDNA Reverse Transcription Kit (High-Capacity cDNA Reverse Transcription Kit, Applied Biosystems™). For each sample, 1 pg of RNA was utilized and reverse- transcribed using 10 pl of cDNA synthesis buffer, 2 pl of reverse transcriptase and a total of 8 pl of sample RNA and DEPC-treated water. The synthesis of cDNA was done in a thermocycler (Biometra T3000 Thermocycler, Biometra, Analytik Jena, Jena, Germany). After cDNA synthesis, 1 pl of E. coli RNase H was added to each sample and placed back into the thermocycler.
Primers and housekeeping genes
To normalize resulting Ct values from the qPCR results, housekeeping genes were used. For the KMR46 samples, mouse GAPDH and HPRT were used as housekeeping genes. For the RD and RD18 samples, GAPDH and RPL13a were used. For both KMR46, RD and RD18, 41 primers were used, including: ACTA1, CDK4, CDK6, MET, MSTN, MYF5, MYH3, MYOD, MYOG, PGK1, PDK1, TNNI1, TNNT1, TNNI2, TAG LN, SDHD, NRAS, MYH8, KLF6, KLF4, EGF, MICAL2, LDHA, IMPDH2, IGFBP2, IGFBP3,
H0XA11, HMGA2, FGFR4, CNN1, CD44, CAV2, CALD1, ALDH1L2, THY1, TGFB1,
SNAI1, PTEN, MYLK, MYBL2 and MEF2C. All primers were synthesized and purchased from Integrated DNA Technologies (IDT, Coralville, IA, USA).
For the master mix, SYBR Green (Thermo Scientific™) was mixed with 1:500 ROX reference dye (Thermo Scientific™). Primer solution was prepared: for each pl of primer mix (forward primer (2.5 pM) + reverse primer (2.5 pM)), 5 pl of master mix was added. Then, for each well of the 384-well plates (4titude® Ltd., FrameStar® 384 Well Skirted PCR Plate, Surrey Hills Business Park, Damphurst Lane, Wotton, Dorking RH5 6QT, United Kingdom (UK)), 5 pl of sample cDNA and 6 pl of the primer solution were added. After all samples were loaded, the plates were sealed and centrifuged (Eppendorf Centrifuge, Model 5702, Eppendorf, Eppendorf, Hamburg, Germany). Finally, RT-qPCR was done using an Applied Biosystems VHA7 machine (Applied Biosystems™, Thermo Scientific™). Data was collected and analyzed using GraphPad Prism 8 software (GraphPad Software Inc., San Diego, CA, USA).
Western blot
Protein isolation
Depending on the size of cellular pellets, pellets were lysed in either 60 pl or 90 pl of RIPA buffer (Thermo Scientific™, #89900). The solutions were sonicated twice for 10 seconds at an amplitude of 10% (Sonifier® Cell Disruptor, Branson Ultrasonics Corporation, Danbury, CT, USA), incubated at room temperature for 10 minutes and centrifuged at 16000 rpm, for 15 minutes at 4°C. The supernatants were collected and quantitatively measured by using the NanoDrop spectrophotometer (NanoDrop® ND-1000, Isogen life science B.V., De Meern, NL). For each thermocycler eppendorf, 40 pg of sample protein was mixed with 5 pl of loading buffer and then boiled at 95°C for 10 minutes in the thermocycler.
Western blot
Western blot analyses were done by using 15% acrylamide hand-cast gels (running gel: Milli-Q water, 30% acrylamide mix (Bio-Rad Laboratories, Inc., Hercules, CA, USA), 1.5 M Tris-HCI (pH 8.8), 10% SDS, 10% ammonium persulfate, 5% TEMED (Merck Millipore), stacking gel (5%): Milli-Q water, 30% acrylamide mix, 1.0 M Tris- HCI (pH 6.8), 10% SDS, 10% ammonium persulfate, 5% TEMED). After loading the ladder (Novex™ Sharp Pre-stained Protein Standard (on gel)/MagicMark™ XP Western Protein Standard (on membrane), Invitrogen™) and the sample, the gel was run at 120V (PowerPac™ Basic Power Supply, Bio-Rad Laboratories, Inc.) until the dye front was close to the end of the gel. Then, the proteins were electro-transferred onto a nitrocellulose membrane with transfer buffer, at 200 amperes for 2 hours and 30 minutes. All of the equipment was purchased from Bio-Rad Laboratories, Inc. (Mini-PROTEAN Tetra Cell). The membranes were then stained with Ponceau (Sigma- Aldrich), to visualize proteins and to check if the transfer was successful. Afterwards, the membranes were washed with TBS-Tween (Thermo Scientific™) and were blocked with 5% (w/v) skim milk (Sigma-Aldrich) in TBS-Tween for one hour at room temperature. After being washed with TBS-Tween twice for 10 minutes on the shaker, the membranes were incubated with primary antibody in 2.5% (w/v) skim milk in TBS-Tween overnight at 4°C. On the second day, the primary antibodies were recollected, and then membranes were washed 3 times with TBS-Tween for 5 minutes. Membranes were incubated with secondary antibody in 2.5% (w/v) skim milk in TBS- Tween for one hour at room temperature.
Visualization of the proteins was done by using a Bio-Rad UV machine (ChemiDoc XRS+ Gel Doc™ XR+ System, Bio-Rad Laboratories, Inc.). To develop and to amplify the signal, chemiluminescent horseradish peroxidase (HRP) substrates, Pico and Femto reagents (SuperSignal™ West Femto Maximum Sensitivity Substrate and SuperSignal™ West Pico PLUS Chemiluminescent Substrate, Thermo Scientific™) were added on top of the membranes, and images were taken immediately afterwards.
Antibodies used for western blot
For the loading controls, GAPDH (rabbit) and alpha-tubulin (mouse) (Sigma Aldrich, G9545, T5168) 1: 1000 in 2.5% (w/v) skim milk in TBS-Tween were used for both RD18 and KMR46. The membranes were first incubated with primary antibody in 2.5% (w/v) skim milk in TBS-Tween overnight at 4°C. The primary antibodies used were 1:500 MYF5 (rabbit) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA, sc- 302), and 1 :3 MYOG (mouse). And the day after incubated with primary antibody, the membranes were incubated with (1:3300 - in 2.5% (w/v) milk in TBS-Tween) anti-rabbit secondary antibody (Abeam, ab6721) (Cell Signaling Technology, #7074) and (1:3300 - in 2.5% (w/v) milk in TBS-Tween) and anti-mouse secondary antibody (Abeam, ab97046).
Western blot quantification
The western blot results were quantified by using Image Studio Lite software (Image studio™ Lite, LI-COR®, Nebraska, USA). EV isolation and analysis
Cell culture
EVs were extracted from the LHCN-M2 (human immortalized myoblasts) cells. First, cells were cultured in medium (1 vol. 199 medium, 4 vols. DMEM, 20% FBS and 50 |jg/ml of gentamycin) (Gibco™) with growth factors (25 pg/ml fetuin (Promocell, Heidelberg, Germany), 5 ng/ml human epidermal growth factor (PeproTech, Rock Hill, NJ, USA), 0.5 ng/ml fibroblast growth factor-basic (PeproTech), 5 pg/ml insulin (Sigma-Aldrich) and 0.2 pg/ml dexamethasone (PeproTech)) until almost 100% confluency was reached. The cells were left in the differentiation medium (DMEM, 10 pg/ml insulin, and 50 pg/ml gentamycin) for 4 days. Finally, the medium was collected for EV extraction.
EV isolation
The collected medium was centrifuged at 500g for 10 minutes (Eppendorf Centrifuge, Model 5810, Eppendorf), 3000g for 20 minutes (Avanti®J-E high-speed centrifuge, Beckman Coulter life sciences, IN, USA), 12,000g for 20 minutes (Beckman coulter), and then 100,000g for 70 minutes (Optima L-90K Ultracentrifuge, Beckman Coulter). After removal of the supernatant, the pellets were resuspended in 5 ml of PBS, centrifuged again at 100,000g for 70 minutes. Finally, the supernatant was removed, and the pellets were resuspended in 400 pl of PBS and stored at -80°C. All of the centrifugation steps were done at 4°C.
Exosome labelling
PBS was used to dilute the isolated EVs to reach a final volume of 500 pl. The suspension was filtered with a 0.2 pm filter (Corning® syringe filters, CLS431212, Corning). 50 pl of lOx Exo-Red (ExoGlow™ - Protein EV labelling kit, System Biosciences, Palo Alto, CA) were added to the purified EVs, incubated at 37°C for 10 minutes and placed on ice for 30 minutes. Finally, the labelled EVs were placed in a 24-well plate.
Nanosiqht analysis
PBS was used to dilute the isolated EVs with a dilution factor of 1: 5 (i.e., 400 pl of EVs in 1.6 ml of PBS), and the suspension was filtered with a 0.2 pm filter. The purified EV samples were then loaded into the sample chamber of a Nanosight machine (NanoSight LM10, Malvern Panalytical Ltd, Malvern, UK) and the quantification and size measurement were done by using the Nanosight NTA 2.3 software (Malvern Panalytical Ltd).
Statistical analysis
All the statistical analyses were done by using GraphPad Prism 8 software. Depending on the data and the analysis purpose, two-tailed t tests or ANOVA tests were done, to see if there are any significant differences between the samples. The confidence level was set at 95%, where probability values of p<0.05 were considered as being statistically significant.
Example 2. Selection of oncosuppressor miRNAs
The miRNA signature was determined of patients diagnosed with embryonal RMS. Data from Bersani et al. (2016) Cancer Res. 76, 6095-6106. (accession number ID PRJNA326118) were used to identify miRNAs not expressed in FN-RMS patients. The predicted genes targeted by the downregulated miRNAs were cross-checked and identified the pathways using g: Profiler. These pathways mainly involved include cell cycle, hippo signalling, fatty acid metabolism and central carbon metabolism. The combination of the key microRNAs that could be used as novel therapeutic agents was identified by checking the single pathways targeted by the microRNAs using DIANA software, and miR-449a and miR-340 were identified as the two key microRNAs in the downregulated microRNA pool.
Example 3.miR-449 and miR-340 dramatically affect tumourigenicity of FN- RMS
The role of the downregulated miRNAs in FN-RMS was investigated, by transfecting both human and mouse in vitro FN-RMS models with miR-449a and miR-340 and subsequently checking the perturbation effects at the transcriptome level with RNAseq analysis. Both the principal component analysis and a volcano plot showed a remarkable difference in miRNA-treated cells compared to vehicle-treated ones. By checking the pathways that are differentially expressed, the downregulated genes are mainly involved in cell cycle, while among the top upregulated pathways in miRNA-treated samples cilium organization and cilium assembly were identified. This is in line with previously reported data regarding quiescent muscle cells that upon exit from the cell cycle undergo cilium formation.
The use of oncosuppressor miRNAs was tested and the potential effects in vitro and in vivo were checked. At the proliferation level, there was a reduction in proliferation in FN-RMS treated cells (figure 6A). To assess whether this effect was due to a potential toxicity of the miRNAs, the effects on proliferation were checked in an in vitro hybrid system by co-culturing FN-RMS and mesoangioblasts (MABs) (Figure 6B). After transfection there was no tangible effect in terms of reduced number of cells in healthy skeletal muscle cells but solely in FN-RMS, which further confirmed the hypothesis of the specificity of these miRNAs in targeting the tumorigenic apparatus of FN-RMS. In order to address whether the reduction in proliferation would coincide with the cell cycle exit identified from the RNAseq, EdU analysis was performed on FN-RMS treated with either 449a/340 or the previously described combination of miR-181a+212 (selected promyogenic cocktail, s-PMC). Compared to the s-PMC, the cells treated with miR-449a+340 showed a complete shift from the S phase into the G0/G1 phase (Figure 6C). As this raised concerns over the possibility that the novel phenotype coincided with an increased potential of cancer sternness, the capability of miRNA-treated cells to form spheroids was explored (Figure 6D). The spheroid diameter in miRNA-treated cells was reduced compared to vehicle-treated one, thus excluding the possibility of an increased presence of cancer stem cells.
Pre-treated murine FN-RMS cell lines were treated using the miRNA combination, and subsequently injected the treated cells in C57BI/6 mice (Figure 6E). After 15 days, the reduction in proliferation of miRNA-treated tumour cells after a single treatment was still statistically significant compared to vehicle-treated implanted cells (Figure 6F). Finally, as the main issue with respect to FN-RMS involves the metastasis to the lungs, the question arises whether RNA-treated tumour cells would have a reduced capacity at migrating compared to vehicle-treated ones. Indeed, both in vitro (Figure 6G) and in vivo (Figure 6H) analysis showed a reduced migration potential of miRNA- treated cells.
Example 4. miR-449a+340 and mitochondrial pyruvate carrier (MPC) inhibition similarly affect proliferation and metastasis
A metabolomic analysis in miRNA-treated FN-RMS cells was performed. At the intracellular level (Figure 7A) An increased accumulation of Valine, Leucine, Isoleucine, Proline, Serine, Methionine, Glutamic acid, Tyrosine, Histidine, Citric acid, Pyruvate, and Phenylalanine was observed. Conversely, a decreased concentration of Fumaric acid, Malic acid and Threonine was measured in miRNA- compared to vehicle-treated FN-RMS cells. By cross-checking the metabolomic data with the transcriptomic data, several pathways were identified that further hint at a switch of the FN-RMS towards myogenic phenotype. Specifically, there was an involvement of pathways being downregulated in FN-RMS patients compared to skeletal muscle (Figure 7B): alanine, aspartate and glutamate metabolism; pyruvate metabolism; citrate cycle; beta-alanine metabolism; valine, leucine and isoleucine degradation. miRNA-treated samples were found to have an increased secretion of alanine (Figure 7C) as well as a switch from glucose uptake to secretion (Figure 7D). The role of pyruvate, was studied at the RNAseq level and it was found that both genes encoding for mitochondrial pyruvate carrier (MPC) were downregulated in miRNA-treated FN-RMS (Figure 8A). By checking the metabolic effects, a reduction in the oxygen consumption rate was observed in human FN-RMS after miRNA treatment, which was similarly observed using MPC inhibitor (Figure 8B). In order to gain a better understanding on the effects of inhibiting MPC in FN-RMS, the effects of the inhibitor on the cell cycle were checked and a similar cell cycle exit was observed after using MPCi (Figure 8C) which was reflected on the proliferation of cells after MPCi or miRNA treatment (Figure 8D). The reduction in cell number could be connected to the increase in size of nuclei after either MPCi or miRNA treatment at day 5 of treatment (Figure 8E). The fusion percentage of cells increased dramatically in both MPCi and miRNA treatment (Figure 8F).
The effects of MPC inhibition in vivo were checked, and a similar reduction in proliferation was observed (Figure 9A-B). Similar to miRNA treatment, the MPCi leads to reduction in metastatic potential of FN-RMS (Figure 9C-D).
To address the effects of MPCi and miRNA treatment on skeletal muscle functionality, the time on treadmill (Figure 9E), distance (Figure 9F) and power (Figure 9G) were checked. Focusing on the time of run, for all the days the vehicle group ran significantly less time than the sham group. The MPCi group ran significantly more time than the vehicle group on day 7 and 11 after the injection. For the miRNA group, the time of run was significantly (p < 0.001) greater than the non-treated group for day 11 and 13. In terms of distance, the results were in line with the ones obtained for the time parameter. The power (W) that the mice exerted in the treadmill was also evaluated. The vehicle mice had significantly less power than the sham group on day 9 and 13. The MPCi mice performed significantly greater than the vehicle mice in terms of power on day 7, 9 and 13. For the mice that received miRNA-treated cells, the power exerted was significantly greater than the one exerted by the vehicle mice on all days. Taken together, on a short-term performance, MPCi and miRNA treated tumour bearing mice were able to perform more similarly to sham mice, in contrast to the poor performance of vehicle mice, affected by the tumour. At the protein level reduced levels of MYOG were found both after miRNA and MPCi treatment (Figure 9H). miRNA modulates and reverses the tumorigenic phenotype at the epigenetic level.
In order to gain a better insight on the regulation mediated by oncosuppressor microRNAs, the effects at the epigenetic level were explored. By analysing the RNAseq data a downregulation of the chromatin modifiers such as PRC1, EZH2 and DOTI L was observed (Figure 10A) which hinted at the possibility of a hypomethylated state upon miRNA treatment. Indeed, educed levels of H3K27me3 (Figure 10B) were observed. Thus, ATACseq in miRNA-treated samples was performed and an increased accessibility to chromatin of genes that are also found to be upregulated at the transcriptome level was observed. By analysing genes using i-cisTarget, PBX1 was identified as the transcription factor with the highest number of enriched features (Figure 5.7D-E). Analysis of single-nuclei RNAseq (snRNAseq) shows upregulation of myoblast feature CREB5, as well as senescence feature OXR1, in miRNA-treated FN- RMS
Example 5. Identification of a novel synergistic combinatorial treatment regimen for FN-RMS
Among the genes found to be downregulated after miRNA perturbation, EZH2, TOP2A and MYBL2 were further investigated. These genes were found to be upregulated in FN-RMS patients compared to skeletal muscle, and they are downregulated by the miRNA combination in FN-RMS. the effects of drugs targeting these genes either alone or in combination were investigates. Tazemetostat (EZH2 inhibitor), Doxorubicin (TOP2A inhibitor) and Topotecan (MYBL2 inhibitor) were determined using XTT proliferation assay which assesses cell viability as a function of redox potential. The use of Tazemetostat alone did not have effects, while at 96h Doxorubicin reduced the activity to 22.8% and Topotecan to 62.1% on FN-RMS cells compared to DMSO. As these targets are downregulated at the same time with miRNA treatment, the effect of these drugs combined was tested. Tazemetostat synergistically worked with Doxorubicin (18.9%) more than with Topotecan (42.3%) in reducing cell activity, while Doxorubicin combined with Topotecan has a dramatic effect on cell proliferation (2%). However, the synergy of the three drugs together has the most impressive effect, with activity being reduced to 0.4% compared to vehicle-treated cells.
Example 6. MPCi-treated cells show similar effects as mi NA-treated samples when looking at the morphology of the cells
In order to investigate the morphological effects of MPCi on human and murine FN- RMS, immunofluorescence staining was done on both murine KMR46 cells and on human RD18 cells, with Hoechst staining the nuclei and phalloidin staining the actin filaments. For both KMR46 and RD18 cells, the staining results showed that there was a lower cell confluence in the MPCi-treated group compared to the DMSO/control group, and an even more significant decrease in the miRNA (hsa-miR-449a-5p and hsa-miR-340-5p)-treated group. Furthermore, compared to the other two sample groups, the MPCi-treated cells were more elongated. Conversely, in the miRNA- treated group, an enlargement of the cells was evident and the cells formed multinucleated structures.
Example 7. MPCi-treated cells show similar effects as miRNA-treated samples in number, size, and multinucleation in the murine KMR46 cell lines When looking at the molecular level of different sample groups, three aspects were evaluated. First, the number of nuclei on day 3 and day 5 of different groups were compared. This was done to check if there was any effect on the proliferation rate of tumour cells after treatment with MPCi or miRNAs. The results showed that on day 3 there was already a decrease in the proliferation rate of the MPCi-treated group compared to the DMSO group. On day 5, the reduction in the rate of proliferation was even more significant (P<0.0001) in the MPCi-treated samples. When the number of nuclei was compared between day 0, day 3 and day 5 of the miRNA- treated group, the results showed a decrease in number of nuclei which indicated cell death instead of a decrease in proliferation. Then, when looking at the changes in the nuclei size, for the DMSO group the nuclei size remained constant on day 3 and day 5 and were relatively smaller than the other two groups. For the other two groups, the same trend of an increase in the size of nuclei was observed.
For the fusion of cells, the calculations were done by looking at the MF20 (Myosin heavy chain) stained cells. Fusion was calculated by using the number of nuclei (two or more) in MF20 stained cells divided by the total number of nuclei in the same field. The results indicated a higher formation of multinucleated cells in both MPCi- and miRNA-treated groups, with the miRNA-treated group showing high incidence of multinucleation at both day 3 and day 5. The MPCi-treated group showed a lower incidence of multinucleation compared to the miRNA group, however on day 5 there was still quite a significant increase in multinucleation compared to the DMSO group. As a result, both MPCi and miRNA treatment could influence the fusion of nuclei in tumour cells, with miRNA treatment having more significant effects.
Example 8. Western blot results show changes of MYF5 and MYOG protein contents after MPCi- and miRNA-treatment
To further investigate the results obtained from the RT-qPCR, western blot was done to check on the contents of MYF5 and MYOG proteins. For the KMR46 samples, the changes of MYF5 protein contents were not significant between treated and untreated samples, and the changes in MYOG protein contents was only significant (*p<0.05) between MPCi- and miRNA-treated samples (Figure 3A and Figure 4A). For the RD18 samples, changes of MYF5 protein content were only significant (*p<0.05) between the DMSO and miRNA-treated samples. Since there were no MYOG protein found in both MPCi- and miRNA-treated samples, the changes of protein contents were significant (**p<0.01) between DMSO and MPCi-treated samples, and between DMSO and miRNA-treated samples (Figure 3B and Figure 3B). GAPDH was used as loading controls, in both KMR46 and RD18 samples GAPDH was reduced in miRNA- treated samples. In KMR46 samples, GAPDH proteins were also reduced in the MPCi- treated samples (Figure 3).
Example 9. EVs analysis
Exosome labeling
The exosomes isolated from the LHCN-M2 cells were visualized by using Exo-Red and checked under the microscope. The results show the presence of exosomal clusters at lOx magnification.
Nanosight was used for quantification of EVs and evaluation of the particle diameter. The results showed EVs isolated from the LHCN-M2 cells had an average size of 143 +/- 92 nm, with a concentration of 0.14 +/- 0.13 E8 particles/ml (Figure 5).

Claims

33 CLAIMS
1. Mir-340 and mir-449a for use as a medicament.
2. Mir-340 and mir-449a for use as a medicament according to ciaim 1, wherein mir-340 is hsa-mir340-5p and mir-449a is hsa-mir-449-5p.
3. Mir-340 and mir-449a for use in the treatment or prevention of a tumour.
4. Mir-340 and mir-449a for use in the treatment or prevention of a tumour according to ciaim 3, in a human individual.
5. Mir-340 and mir-449a for use in the treatment or prevention of a tumour according to claim 3 or 4, wherein the tumour is a soft tissue sarcoma, such as a paediatric soft tissue sarcoma.
6. Mir-340 and mir-449a for use in the treatment or prevention of a tumour according to any one of claims 3 to 5, wherein the tumour is rhabdomyosarcoma.
7. Mir-340 and mir-449a for use in the treatment or prevention of rhabdomyosarcoma (RMS) according to claim 6, wherein the rhabdomyosarcoma is selected from the group of embryonal RMS, alveolar RMS, spindle cell RMS, mixed-type RMS, pleomorphic RMS, and RMS with ganglionic differentiation and alveolar RMS.
8. Mir-340 and mir-449a for use in the treatment or prevention of rhabdomyosarcoma (RMS) according to claim 6 or 7, wherein the rhabdomyosarcoma is fusion-positive RMS (FP-RMS) or fusion-negative RMS (FN-RMS).
9. Mir-340 and mir-449a for use in the treatment or prevention of a tumour according to any one of claims 3 to 6, wherein mir-340 is hsa-mir340-5p and mir-449a is hsa-mir-449-5p. 34
10. Mir-340 and mir-449a for use in the treatment or prevention of a tumour according to any one of claims 3 to 9, wherein the Mir are administered by extracellular vesicles (EVs) or exosomes.
11. A pharmaceutical composition comprising mir-340 and mir-449a.
12. The composition according to claim 11, wherein mir-340 is hsa-mir340-5p and mir-449a is hsa-mir-449-5p.
13. The composition according to claim 11 or 12, which is formulated in extracellular vesicles (EVs) or exosomes.
14. A method of treating tumour in an individual comprising the step of administering mir-340 and mir-449a to said individual, thereby converting tumour or tumorigenic cells into cell with a non-tumorigenic phenotype.
15. The method according to claim 14, wherein the tumour is rhabdomyosarcoma.
16. The method according to claim 14 or 15, wherein mir-340 is hsa-mir340-5p and mir-449a is hsa-mir-449-5p.
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