Therefore, its down-modulation at later time points may be needed in order to trigger cell death. Interestingly, our data on ��-catenin modulation are in contrast to the original report [25] showing that pyrvinium pamoate induced its degradation in APC-mutant cells (SW480) but not in ��-catenin-mutant cells (HCT-116 WTKO). We found that ��-catenin was slightly down-regulated kinase inhibitor ARQ197 in Ls174T cells, which express a non-phosphorylatable mutant form of ��-catenin, but not in DLD-1 cells, expressing wild-type ��-catenin. Thus, the effects on ��-catenin expression do not seem to correlate with its genetics. More studies are needed to address this point. Clearly, however, the growth inhibitory activity of pyrvimium correlates with down-regulation of pygopus.

In conclusion, this study confirms and extends our previous data showing that concomitant inhibition of two commonly mutated pathways (Wnt and KRAS) leads to superior antitumor effects in colon cancer cells that carry those mutations. The synergistic activity was specific, as it was not observed in CRC cells expressing wild-type KRAS. The potential advantages offered by a synergistic combined treatment resides in the possibility to (i) shut down both oncogenic drivers and (ii) reduce drug doses, thus limiting toxic effects. The obvious limitation of this work is the lack of in vivo validation. Unfortunately, the effects of these drug combinations could not be evaluated in mice for technical reasons. Despite this, in our opinion, the in vitro results presented here represent a convincing proof-of-principle for double pharmacologic targeting and encourage the development of clinical ��-catenin and KRAS inhibitors for targeted cancer therapy.

Supporting Information Figure S1 Effects of PKF115-584, Pyrvinium and FTS treatments in DLD-1 cells. The cells were treated with increasing concentrations of PKF115-584 (A, D), pyrvinium (B, E) or FTS (C, F). Cell growth was measured at 72 hours Drug_discovery by MTS assay (A�CC). Total lysates were probed with the indicated antibodies (D�CF). (TIF) Click here for additional data file.(860K, tif) Figure S2 Sequence of the N-terminal b-catenin region in DLD-1 and Ls174T cells. The known CK1�� and GSK3�� target aminoacids 33, 37, 41 and 45 are indicated. Mutation of Ser45 to Phe is confirmed in Ls174T cells. (TIF) Click here for additional data file.(452K, tif) Figure S3 Effects of PKF115-584/PLX-4032 combination in HT-29 cells. (A) The cells were cultured for 6 days in the presence of inhibitors as single agents or in combination (PKF =0.5 ��M; PLX =2 ��M). Percent growth was evaluated by MTS assay. (B) Combination Index values were calculated by CalcuSyn. (TIF) Click here for additional data file.