Abstract IA05: Novel function of microRNAs.

MicroRNAs (miRNAs) are small noncoding RNAs, 19-24 nucleotides in length, which regulate gene expression, and are aberrantly expressed in most types of cancer. MiRNAs have also been detected in the blood of cancer patients, and can serve as circulating biomarkers. It has been shown that secreted miRNAs within exosomes can be transferred from cell to cell, and can regulate gene expression in the receiving cells by canonical binding to their target messenger RNAs. Here we show that tumor secreted miR-21 and miR-29a can also function by a novel mechanism, by binding as ligands to receptors of the Toll-like receptor family, murine TLR7 and human TLR8, in immune cells, triggering a TLR-mediated prometastatic inflammatory response, which ultimately may lead to tumor growth and metastasis. Thus, by acting as peregrine agonists of TLRs, secreted miRNAs are key regulators of the tumor microenvironment. This mechanism of action of miRNAs is implicated in the tumor-immune system communication, and is important in tumor growth and spread, therefore representing a new possible target for cancer treatment. The cachexia syndrome is a debilitating state of cancer that predominantly results from the loss of skeletal muscle mass, which is in part associated with apoptosis. How tumors promote apoptosis in distally located skeletal muscles has not been explored. Using both tumor cell lines and patient samples, we show that tumor-derived microvesicles induce apoptosis of skeletal muscle cells. This pro- apoptotic activity is mediated by a microRNA cargo, miR-21, which signals through the Toll-like 7 receptor (TLR7) on murine myoblasts to promote cell death. Furthermore, tumor microvesicles and miR-21 require c-Jun N-terminal kinase activity to regulate this apoptotic response. Together, these results describe a unique pathway by which tumor cells promote muscle loss, which might provide a great insight into elucidating the causes and treatment options of cancer cachexia. Apoptosis has been associated with cancer cachexia. However, it is not clear so far as to the signals inducing cell death and the type of cells undergoing apoptosis. Our results indicate that circulating MVs secreted by mouse and human cancer cells cause apoptosis of muscle cells, and that this phenomenon is dependent on TLR7 (mouse) or TLR8 (human). Importantly, cancer cell lines that are usually associated with cancer cachexia, such as lung cancer and pancreatic cancer cell lines, were able to induce myoblast cell death, but not breast cancer cell lines. These results suggest the specificity of MV-mediated cell death in the context of muscle wasting associated with cancer cachexia. We further found that miR-21 levels secreted into the MVs were elevated in those cell lines that induced muscle cell death. One of the pancreatic cell lines, PC1, induced the strongest effect of cell death and contained the highest levels of miR-21 in the MV cargo. Inhibition of miR-21, a ligand of TLR7 and TLR8, inhibited the induction of apoptosis of the muscle cells. Thus, on the basis of these findings, we predict that MVs secreted by cancer cells overexpressing miR-21, similar to those derived from pancreatic and lung malignancies, fuse with muscle cells and induce apoptosis by activating TLR7/8. One interesting observation is that cancer cell lines that induce muscle cell death also seem to secrete more MVs), which contributes to the phenomenon of a higher level of miR21 expression. Future studies will investigate whether a higher number of MVs is associated with the deregulation of other miRNAs or signaling pathways, as blocking TLR7 does not completely rescue miR21-induced cell death. Taken together, our results provide insights into therapeutic avenues for cachexia, possibly by inhibiting MV secretion, inhibiting fusion of MV with muscle cells, or blocking the binding of miR-21 to TLR7/8, which are currently under investigation. Citation Format: Carlo M. Croce. Novel function of microRNAs. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr IA05.