Final Report Summary - MIRNATHERAPYNBL (New epigenetic therapy for high-risk neuroblastoma)
Despite multimodal therapies, a high percentage of high-risk neuroblastomas (NBs) become refractory to current treatments, most of which interfere with cell cycle and DNA synthesis or function, activating the DNA damage response (DDR). In cancer, this process is frequently altered by deregulated expression or function of several genes which contribute to multidrug resistance (MDR).
MicroRNAs (miRNAs) are endogenous non-coding small RNAs that interfere with the translation and stability of coding messenger RNAs (mRNAs) in a sequence-specific manner. In recent years, miRNAs have been found to be deregulated in a number of different human cancers, including NB, and the over- or under- expression of specific miRNAs correlates with stage, progression and patient outcome. Therefore, the alteration in miRNA levels may contribute to the functional behavior of high-risk NB, including its resistance to multiple drugs. MiRNAs are outstanding candidates for therapy since a single microRNA can modulate the expression of multiple genes of the same or different pathways, thus hindering the development of resistance mechanisms by the tumor.
The working hypothesis of this grant is that miRNA restoration represents an attractive novel therapeutic approach for the treatment of high-risk NB. The main goal of this project is finding the miRNA(s) with the highest therapeutic potential through the following aims:
Aim 1. Functional screening of miRNAs in chemoresistant NB cell lines.
Aim 2. Analysis of the miRNA downstream targets.
Aim 3. Development of microRNA-delivery system to target Neuroblastoma cells in vivo.
To identify tumour-suppressive miRNAs we performed a high-throughput functional screening of 2048 miRNAs. Among those miRNAs found to reduce cell proliferation, we deeply characterized the therapeutic potential of those that are located in the chromosomic region 14q32, a genomic locus frequently altered in cancer. MiRNAs effects on proliferation and viability were analyzed by FACS and western-blot. MiRNA-target analysis was performed in silico and validated by quantitative real-time PCR, western blot and luciferase-reporter assays. The therapeutic potential of miRNAs was further evaluated in mouse preclinical xenograft models.
In summary, we have identified and characterized at least three different tumour-suppressive miRNAs in NB. The ectopic expression of these miRNAs reduces cell proliferation and viability in multiple chemoresistant NB cell lines in vitro and in vivo through the modulation of several cell cycle and survival genes.
Furthermore, we have set up the procedures to prepare multifunctional nanovesicle-miRNA conjugates using a simple one-step methodology, named DELOS and based on the use of CO2-expanded solvents. This System allows to prepare highly homogeneous and reproducible suspensions of unilamellar nanovesicles that can encapsulate small RNA molecules such as miRNAs
Conclusions: MicroRNA-based restoration therapies are a therapeutic alternative against neuroblastomas resistant to conventional therapies.
MicroRNAs (miRNAs) are endogenous non-coding small RNAs that interfere with the translation and stability of coding messenger RNAs (mRNAs) in a sequence-specific manner. In recent years, miRNAs have been found to be deregulated in a number of different human cancers, including NB, and the over- or under- expression of specific miRNAs correlates with stage, progression and patient outcome. Therefore, the alteration in miRNA levels may contribute to the functional behavior of high-risk NB, including its resistance to multiple drugs. MiRNAs are outstanding candidates for therapy since a single microRNA can modulate the expression of multiple genes of the same or different pathways, thus hindering the development of resistance mechanisms by the tumor.
The working hypothesis of this grant is that miRNA restoration represents an attractive novel therapeutic approach for the treatment of high-risk NB. The main goal of this project is finding the miRNA(s) with the highest therapeutic potential through the following aims:
Aim 1. Functional screening of miRNAs in chemoresistant NB cell lines.
Aim 2. Analysis of the miRNA downstream targets.
Aim 3. Development of microRNA-delivery system to target Neuroblastoma cells in vivo.
To identify tumour-suppressive miRNAs we performed a high-throughput functional screening of 2048 miRNAs. Among those miRNAs found to reduce cell proliferation, we deeply characterized the therapeutic potential of those that are located in the chromosomic region 14q32, a genomic locus frequently altered in cancer. MiRNAs effects on proliferation and viability were analyzed by FACS and western-blot. MiRNA-target analysis was performed in silico and validated by quantitative real-time PCR, western blot and luciferase-reporter assays. The therapeutic potential of miRNAs was further evaluated in mouse preclinical xenograft models.
In summary, we have identified and characterized at least three different tumour-suppressive miRNAs in NB. The ectopic expression of these miRNAs reduces cell proliferation and viability in multiple chemoresistant NB cell lines in vitro and in vivo through the modulation of several cell cycle and survival genes.
Furthermore, we have set up the procedures to prepare multifunctional nanovesicle-miRNA conjugates using a simple one-step methodology, named DELOS and based on the use of CO2-expanded solvents. This System allows to prepare highly homogeneous and reproducible suspensions of unilamellar nanovesicles that can encapsulate small RNA molecules such as miRNAs
Conclusions: MicroRNA-based restoration therapies are a therapeutic alternative against neuroblastomas resistant to conventional therapies.