In addition, up-regulation of the pro-apoptotic protein APAF1 was noted. Interestingly, p21WAF1 was transiently up-regulated at 24 hours before being down-regulated at 72 hours. To further confirm these data, a few genes were analysed independently by standard real-time PCR in four cell lines. Figure 4B shows read this the results from Ls174T cells, confirming gene regulation observed in the original data set. The data from all four cell lines were further analysed to identify synergistic gene expression modulation, defined as >2-fold change of expression in a combination compared to single treatments (figure 4C). Interestingly, BCL2 expression was synergistically down-regulated in all four cell lines by pyrvinium/FTS combination (filled grey boxes). Cyclin D1 and CD44 scored synergistic in three out of four cell lines.
CD44 was also down-regulated in DLD-1 cells, but the extent of modulation did not reach the threshold for a synergistic effect (figure S8). The PKF115-584/FTS combination proved less effective in this analysis, with only CD44 showing synergistic down-regulation in at least two cell lines (Ls174T and SW480). These results indicate CD44 as a common response to the double ��-catenin/KRAS targeting. Figure 4 Expression analysis of selected genes after inhibitors treatments. Having proved the efficacy of double Wnt/KRAS inhibition in vitro, we tried to translate these findings into a novel therapeutic strategy in vivo. However, PKF115-584 is no longer produced by Novartis and its synthesis in large scale proved extremely challenging and time-consuming.
As for pyrvinium pamoate, it is reported to be poorly absorbed [47]. However, oral administration of pyrvinium was described by two groups [48], [49]. Therefore, a pilot experiment was run to verify if the drug could reach the target in nude mice, by looking at pygopus expression in Ls174T xenografts after three daily oral administrations of 10 mg/kg pyrvinium. The results were negative (figure S9), therefore we did not attempt further in vivo analyses. Discussion Almost all colorectal cancers show alteration of the Wnt pathway that controls ��-catenin activation. Therefore, inhibition of ��-catenin may lead to significant progress in the management of this disease. However, recent data revealed that more than one ��driver�� oncogenic pathway is often activated in a single tumor [50], [51].
Therefore, multiple oncogene targeting is likely to be needed to eradicate the disease. In a significant fraction of colorectal tumors, Wnt and KRAS pathway alterations coexist [6], [13], [14]. Despite the fact that it has been known for nearly 30 years, the KRAS oncogene has been an elusive target so far. Agents AV-951 that block KRAS post-translational modifications have been ineffective in clinical trials [52].