Mir-29a Displays in Vitro and in Vivo Anti-Tumor Activity in Multiple Myeloma

Despite advances in recent therapeutic approaches including targeted therapies, multiple myeloma (MM) remains still incurable necessitating the development of novel treatment strategies. MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate post-transcriptional gene expression and play a critical role in tumor pathogenesis. Tumor suppressor miRNAs are generally down-regulated in cancer cells compared to their normal counterpart, and their enforced expression indeed represents a promising strategy for cancer treatment. In this study, we sought to characterize the role of miR-29a as a tumor suppressor as well as evaluated its therapeutic potential in MM.

miR-29a expression levels were found down-regulated in a panel of 5 MM cell lines, 6 newly diagnosed MM patient samples compared to its expression in normal hematopoietic cells collected from 10 normal healthy individuals suggesting that high expression of miR-29a might be involved in MM pathogenesis. We further assessed the functional significance of miR-29a by both gain- and loss-of-function studies. A significant decrease in cell viability (22-32%, p<0.05), along with induction of apoptosis (30-35%, p<0.05) was observed at 48 hrs in MM cell lines, MM.1S and 8226 transfected with miR-29a compared to cells transfected with scrambled miRNA. In contrast, cell lines transfected with miR-29a antagonist prevented the loss of viability in such cells indicating the specificity of miR-29a. At the molecular level, we have identified c-Myc, an important oncogenic transcription factor known to stimulate MM cell proliferation, as a target of miR-29a. Binding site of miR-29a was first identified by computer algorithm and further confirmed by the use of a 3’UTR of c-Myc reporter (luciferase renilla/firefly) constructs containing, miR-29a target site. Moreover, treatment with PRIMA-^Met, a small molecule anti-tumor agent in phase I/II clinical trials, significantly increased the expression of miR-29a (2 to 6-fold) and decreased expression of c-Myc in MM cell lines and primay MM patient samples suggesting an important role of miR-29a in inhibiting proliferation of MM cells. On the other hand, overexpression of c-Myc in 8226 and MM.1S cells at least partially reverted the functional effect of miR-29a or PRIMA-1^Metsuggesting a specific role of c-Myc in mediating its anti-proliferative activity.

To examine therapeutic potential of our studies, we took advantage of novel lipid based delivery method of miRNA. Intratumor delivery of the miR-29a by intraperitoneal injection route against MM xenografts in SCID mice resulted in a significant inhibition of tumor growth (~60%) at 12 days of treatment and prolongation of survival (median survival increased from 22 days to 35 days, p<0.038) compared to the mice receiving scrambled miRNA. Retrieved tumors from treated mice showed efficient increase in miR-29a (5.5-fold, p=0.025), and decrease in c-Myc protein as well as reduced expression of Ki67 and increase of Tunel expression. Similar phenomenon was observed by systematic delivery of miR-29a (by intraveneous injection) in mice with no significant side effects or toxicity in mice.

Our study reveals an important role of miR-29a as a tumor suppressor in mediating anti-tumor activities in MM cells by targeting c-Myc. Our findings provide a proof-of-principle that formulated synthetic miR-29a exerts therapeutic activity in preclinical models, and support a framework for development of miR-29a based treatment strategies in MM patients.