Utilización de RNA de interferencia como estrategia para el estudio y tratamiento del cáncer

Abstract

The recent discovery and characterization of RNA interference (RNAi) provides us with a new tool for gene inactivation and cancer gene therapy. Here, we have investigated the effect of knocking down target proteins by short hairpin RNAs (shRNAs) expressed from viral vectors. The senescence process of primary cell cultures occurs after a finite proliferative doublings. In the present study, we tested a new strategy for mouse primary cultures immortalization using a retrovirus carrying the shRNA sequence against the p53 wild type protein. The inhibition of over 80 % of p53 in murine primary cells leads to cell immortalization. We have also constructed retrovirus to inhibit other target proteins to use as potential cancer gene therapy weapons. Cyclin E1 is expressed during the late G1 phase of the cell cycle and mediates the initiation of DNA synthesis by activating cyclin-dependent kinases 2 (CDK2). We found that the knockdown of cyclin E1 in cancer cells triggers considerable apoptotic induction. The correct segregation of chromosomes during the M-phase is under control of numerous proteins and protein subcomplexes localized to kinetochores that monitor the accuracy of this process. Hec1 (highly expressed in cancer) plays an important role in chromosome segregation by interacting with a subset of checkpoint proteins that survey proper chromosome alignment and bipolar spindle attachment. In order to disrupt mitotic progression of tumor cell lines, we have used retroviral and adenoviral vectors that inhibit Hec1 synthesis. Vectorexpressed shRNA caused depletion of the target protein, cellular arrest and considerable mitotic catastrophe induction in cancer cells. Furthermore, adenocarcinomas induced in the flanks of nude mice show significant reduction in size compared with control when treated with either Hec1-shRNA retroviruses or adenoviruses. Finally, we tested the possibility of using RNAi to silence the AP-1 transcription factor, in particular the Jun proteins that play critical roles in regulating cell proliferation and tumor progression. Reduction of JunB levels causes increased proliferation and tumorigenicity in wild-type murine fibroblasts, whereas in c-Jun knockout cells p53-independent cell cycle arrest and apoptosis are induced. Using melanoma-derived B16-F10 cancer cells the combination of JunB knockdown and c-Jun/JNK inactivation leads to cell cycle arrest and AIF-dependent apoptosis. Furthermore, the combined treatment extends survival of mice inoculated with the tumor cells.