Micro-RNAs in ovarian cancer as tools for diagnosis and therapy /

Abstract

Ovarian cancer (OC) is the fifth cause of cancer in women and the leading cause of death among gynecological malignancies in developed countries. The unspecific symptoms and currently insufficient diagnostic tools fail to detect the disease at an early stage, when the 5-years survival is 90%. Thus, around 70% of the patients are diagnosed at late stage, when the 5-years survival is <25%. Also, while 80% of the patients are initially chemosensitive, 85% of these develop resistance and die of recurrence. Therefore, identifying new diagnostic biomarkers would potentially improve the early detection of OC. Also, developing novel and efficient therapeutic strategies is paramount. Micro-RNAs (miRNAs) are small non-coding RNAs of 22nt that regulate multiple cellular processes by silencing of the specific target mRNAs, and have been found deregulated in cancer, being potential therapeutic elements. In addition, they are stable in circulation therefore being potential non-invasive diagnostic tools by using saliva as source of biomarkers. The first objective was to identify new miRNA diagnostic biomarkers for high-grade serous carcinoma (HGSC). Thus, 32 salivas were subjected for RNA-sequencing, in particular from early- and late-stage HGSC and control patients. Around 100 ng of RNA was found per 1 mL of saliva from control and HGSC patients, which was of sufficient quality for RNA sequencing. First bioinformatic analyses showed around 36% of alignment with the human genome, thereby resulting in a more than 500 known miRNAs and 65 De Novo miRNAs detected on average in the patients’ cohort. Differential expression analysis showed that 49 and 45 miRNAs were significantly deregulated in salivas from early- and late-stage HGSC patients compared to controls, respectively. Interestingly, miR-34 family appeared commonly altered, with three members of the family overexpressed in saliva from HGSC patients, either from early and late-stages, suggesting that they could be potential biomarkers to improve the early detection of HGSC patients, the most fatal subtype of OC. The second objective was to identify new miRNA-based therapies for late-stage OC, since targeted therapies has became the Holy Grail for cancer therapy. In this study, miR-654 was found significantly under-expressed in OC tissues compared to benign ovaries. Overexpression of this miRNA in clinically relevant OC cell lines resulted in a significant decrease in cell proliferation and marked increased apoptotic cell death in vitro, accompanied by an activation of the apoptotic pathway seen at cellular and molecular level. Importantly, ectopic expression of miR-654 reproduced the described phenotypic consequences in vivo. In addition, a pre-clinical model using 4 patient-derived ascitic cells showed that overexpression of miR-654 decreased the sphere forming capacity and reduced spheroid viability. In silico bioinformatics analysis of putative miR-654 targets predicted several genes, among which HAX1, RAB1B, PBX3, CDCP1 and PLAGL2 decreased at mRNA and protein level. A 3’UTR luciferase reporter assay confirmed that miR-654 is a direct of the 5 targets abovementioned. Additionally, silencing of the direct target genes showed that CDCP1 and PLAGL2 depletion phenocopied the effects of miR-654 overexpression, thereby resulting in a reduced proliferation and in an increased apoptosis. Interestingly, both genes were diminished at protein level upon miR-654-5p in the pre-clinical model using patient-derived ascitic cells, suggesting that the therapeutic effect of the miR-654 could be, in part, due to the inhibition of CDCP1 and PLAGL2. Finally, microarray analysis showed that the depletion of CDCP1 and PLALG2 altered MYC, Wnt/β-cat, AKT and MAPK pathways, which has been confirmed by the overexpression of miR-654. Altogether suggested that ectopic expression of miR-654 impaired canonical pathways in OC and that the use of this miRNA as a therapeutic tool might improve the current therapies, potentially in combination with the standard chemotherapy.