MicroRNAs as potential agents to alter resistance to cytotoxic anticancer therapy

Abstract

Tumor cells use preexisting prosurvival signaling pathways to evade the damaging and cytotoxic effects of anticancer agents. Radiation therapy is a primary form of cytotoxic anticancer treatment, but agents that successfully modify the radiation response in vivo are lacking. MicroRNAs (miRNA) are global gene regulators that play critical roles in oncogenesis and have been found to regulate prosurvival pathways. However, there is little understanding of how cellular miRNA expression affects the response of a cancer to cytotoxic therapy and ultimately outcome. The let-7 family of miRNAs regulates expression of oncogenes, such as RAS, and is specifically down-regulated in many cancer subtypes. In fact, low levels of let-7 predict a poor outcome in lung cancer. Here, we report that the let-7 family of miRNAs is overrepresented in a class of miRNAs exhibiting altered expression in response to radiation. More strikingly, we also can create a radiosensitive state when the select let-7 family of miRNAs is overexpressed in vitro in lung cancer cells and in vivo in a Caenorhabditis elegans model of radiation-induced cell death, whereas decreasing their levels causes radioresistance. In C. elegans, we show that this is partly through control of the proto-oncogene homologue let-60/RAS and genes in the DNA damage response pathway. These findings are the first direct evidence that miRNAs can suppress resistance to anticancer cytotoxic therapy, a common feature of cancer cells, and suggest that miRNAs may be a viable tool to augment current cancer therapies.

Figure 1

Relative levels of the let-7 microRNAs change significantly after irradiation. A, miRNA microarrays were performed on total RNA collected from irradiated A549 cells before and 2, 8, and 24 h after 2.5 Gy. The level of each member of the let-7 family changed significantly over this time. B, levels of let-7 miRNAs after irradiation in a normal lung epithelial line, CRL2741. C, real-time PCR results at 8 and 24 h of let-7a, let-7b, and let-7g from a separate A549 replicate and two additional lung cancer cell lines (A427, carcinoma; H441, adenocarcinoma). let-7a and let-7b levels are reduced and let-7g levels are increased after irradiation. Ratio of let-7 levels with the unirradiated 0 time point as the baseline. Bars, SD.

Figure 2

Manipulating miRNA levels alters the radiation response in A549 cells. A, overexpression of let-7b causes significant radiosensitization. A549 cells were transfected with pre-let-7b and irradiated 24 h later with 2.0, 4.0, or 6.0 Gy and then analyzed by clonogenic assay. Results are depicted as dose-response curves. B, overexpression of let-7g causes significant radioprotection in A549 cells. Cells were transfected and irradiated as described above. C, decreasing let-7b causes significant radioprotection in A549 cells. Cells were transfected with anti-let-7b. D, decreasing let-7g causes significant radiosensitivity in A549 cells. Cells were transfected with anti-let-7g and treated as described above. Bars, SD.

Figure 3

Manipulating miRNA levels alters the radiation response in C. elegans. A, overexpression of let-7 and its homologue mir-84 causes radiosensitization in a C. elegans radiation model. Results are depicted as dose-response curves, dose on the X axis and percent WT vulvas on the Y axis. P values are listed next to the curves they represent compared with WT animals. B, loss of the let-7 homologues, mir-84 and mir-48, causes radiation resistance in VPCs. P values represent the mir- strains compared with WT animals. Bars, SD.

Figure 4

RNAi against RAS and DDR genes reverses the mir-84 radiation-resistant phenotype. A, a depiction of how the C. elegans RNAi experiments were performed. Synchronized embryos are placed on plates with the appropriate bacterial strain containing the plasmid that overexpresses dsRNA from the gene of interest and grown until appropriate time for radiation. Radiation is delivered at the previously defined appropriate point in the life cycle of the animal. B, RNAi for RAS and DDR genes leads to reversal of the radiation-resistant phenotype of the mir-84 (KO) strain. Results are normalized to mir-84. Bars, SD. Stratified t tests were performed to evaluate significance, individual dose points that were significant are marked with an asterisk, and P values based on a two-tailed evaluation of the data across both doses were significant for all strains and are as follows: let-60/RAS(RNAi), P = 0.05; rad-51/RAD51 (RNAi), P = 0.05; coh-1/RAD21(RNAi), P = 0.01; fcd-2/FANCD2(RNAi), P = 0.02; and cdc-25.3/CDC25(RNAi), P = 0.03.