Abstract
miR-221/222 are two highly homologous microRNAs (miRNAs), encoded in tandem on the chromosome X, whose up-regulation has been found in several malignancies and are thought to promote cell proliferation via down-regulation of p27 and/or p57, two negative regulators of G1 to S phase cell cycle progression. We demonstrated up-regulation of both miRNAs in malignant plasma cells (PCs) from multiple myeloma (MM) patients belonging to distinct TC (translocation/Cyclin) groups, including TC2 and TC4. A rising body of evidence suggests that silencing miRNAs with oncogenic potential could represent a novel approach for human cancer therapy. We previously demonstrated that silencing miR-221/222 exerts significant anti-MM activity and triggers canonical targets in vitro and in vivo. Here, in the aim to progress to clinical translation of our proof-of-principle findings, we investigated the anti-tumor activity and the appropriateness for systemic delivery of a novel and originally designed LNA-miR-221, a 13-mer antisense miR-221 inhibitor, which took advantage of locked nucleic acid (LNA) technology and phosphorothioate backbone chemistry for increasing affinity for miR-221 and nuclease resistance. We found that enforced ectopic expression of LNA-miR-221 in t(4;14) MM cells significantly inhibited growth and survival of MM cells in vitro. In treated cells, we detected knock down of miR-221/222 together and increased levels of both p27Kip1 mRNA and protein. Specific activity of this LNA-miR-221 inhibitor was confirmed by the use of a 3’UTR reporter (luciferase renilla/firefly) constructs containing, miR-221 target site. This construct was co-transfected either with miR-221/222 mimics or LNA-miR-221 inhibitor into MM cells. As predicted, a reduced luciferase activity was detected in miR-221/222 mimics co-transfected cells with each 3’UTR reporter plasmid, while increase luciferase activity was measured in MM cells co-transfected LNA-miR-221 inhibitors indicating an efficient and stable binding to the miRNA target sequence. Importantly, we evaluated the systemic delivery of the LNA-miR-221 inhibitor with saline solution vehicle alone by intraperitoneal or intravenous injection route against MM xenografts in SCID/NOD mice. Significant anti-tumor activity was achieved after 2 weeks of treatment at similar extent by both injection routes. Retrieved tumors from treated animals showed efficient inhibition of miR-221/222, as demonstrated by increased levels of p27Kip1 protein in vivo. H&E staining and immunohistochemical analysis showed wide necrosis areas, reduced Ki67 and a significant increase of p27Kip1 cytoplasmic expression in retrieved tumors from LNA-miR-221 inhibitor-treated mice. No changes in mice behavior or organ toxicity were observed in treated mice. Taken together these findings support the rationale for development of this novel and highly efficient LNA-miR-221 inhibitor as a promising anti-MM drug in subsequent primate toxicology studies. Supported by the Italian Association for Cancer Research (AIRC), PI: PT. “Special Program Molecular Clinical Oncology - 5 per mille” n. 9980, 2010/15.
Disclosures:
No relevant conflicts of interest to declare.
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