Synergistic effects of combined Wnt/KRAS inhibition in colorectal cancer cells

Abstract

Activation of Wnt signalling due to inability to degrade β-catenin is found in >85% of colorectal cancers. Approximately half of colon cancers express a constitutively active KRAS protein. A significant fraction of patients show both abnormalities. We previously reported that simultaneous down-regulation of both β-catenin and KRAS was necessary to induce significant cell death and tumor growth inhibition of colorectal cancer cells. Although attractive, an RNAi-based therapeutic approach is still far from being employed in the clinical setting. Therefore, we sought to recapitulate our previous findings by the use of small-molecule inhibitors of β-catenin and KRAS. We show here that the β-catenin inhibitors PKF115-584 and pyrvinium pamoate block β-catenin-dependent transcriptional activity and synergize with the KRAS inhibitor S-trans, trans-farnesylthiosalicylic acid (FTS, salirasib) in colon cancer cells driven by Wnt and KRAS oncogenic signals, but not in cells carrying BRAF mutations. The combined use of these compounds was superior to the use of any drug alone in inducing cell growth arrest, cell death, MYC and survivin down-modulation, and inhibition of anchorage-independent growth. Expression analysis of selected cancer-relevant genes revealed down-regulation of CD44 as a common response to the combined treatments. These data provide a proof of principle for a combination therapeutic strategy in colorectal cancer.

Figure 1. PKF115-584, pyrvinium pamoate and FTS activity in Ls174T cells.

(A–C) Chemical structures of PKF115-584, pyrvinium and FTS, as previously described (see ref. 20–29) (D–F) Dose-response effects of PKF115-584, pyrvinium and FTS on Ls174T cells growth. The cells were exposed at increasing doses of each inhibitor for 72 hours. MTS assay was used to evaluate the effect of the compounds on cell proliferation. IC₅₀ values are shown for each compound. (G–H) Luciferase activity from the TOPflash plasmid was determined after incubation for 24 hours with PKF115-584 or pyrvinium. Values are Relative Light Units (RLU) with DMSO-treated cells set as 1.00. (I) Western blot analysis of active GTP-loaded KRAS pull-down (upper panel) and total KRAS (bottom) from Ls174T cells treated with FTS. (J–L) Western blot analysis showing c-myc expression in Ls174T cells treated with increasing concentrations of each compound for 48 hours. From pyrvinium-treated cells, pygopus and β-catenin expression are also shown (K). Actin is always shown as a loading control. (M–N) Quantitative PCR analysis of AXIN2 and CCND1/cyclin D1 expression after treatment with increasing doses (0.125–1.0 μM) of PKF115-584 (M) and pyrvinium (N). (O) Western blot analysis of MEK phosphorylation in FTS-treated cells. Total MEK and actin are shown as controls. (P–Q) Dose-response curves of PKF115-584 and pyrvinium in the absence (empty circles) or presence (filled circles) of 100 μM FTS. Each individual curve is normalized on the corresponding sample with no β-catenin inhibitor.

Figure 2. Synergistic effects of PKF115-584/FTS and pyrvinium/FTS combinations on CRC cells.

The indicated cell lines were cultured for 6 days in the presence of inhibitors as single agents or in combination, or vehicle alone. MTS assay was used to assess the effects of each treatment on cell culture growth and viability. (A) PKF115-584/FTS and (B) pyrvinium/FTS combination. For each cell line, the bar graph (left) shows the effect at a single dose (PKF115-584, 0.25 μM [Ls174T and DLD-1] or 0.125 μM [SW480 and HCT-116]; pyrvinium, 50 nM; FTS, 100 μM). The XY graph (right) shows combination indexes as a function of the fraction affected (see Materials and Methods). For each cell line, the genes that are mutated, affecting Wnt and Ras pathways, are indicated in brackets. CTRL  =  control; Pyrv  =  pyrvinium. In all panels, number symbols (#) indicate significance versus the β-catenin inhibitor (PKF115-584 or pyrvinium); asterisks (*) indicates significance versus FTS; one symbol, p<0.05; two symbols, p<0.01; three symbols p<0.001.

Figure 3. Characterization of synergism in Ls174T cells.

(A) Cells were treated with 100 μM FTS, 0.25 μM PKF115-584 or 50 nM pyrvinium, alone or in combinations, or vehicle, for 6 days. Cell culture viability was assessed on days 3, 4 and 6 by MTS. The relative OD compared to vehicle-treated control cells was recorded as surviving fraction. (B) Ls174T cells were cultured for 3 days in the presence of the indicated compounds (same concentrations as in [A]); in parallel, Ls174T cells carrying inducible siRNAs (siNT, non-targeting; siBC, anti-β-catenin; siKR, anti-KRAS) were treated with doxycycline for the same time. Apoptosis was determined by annexin V/propidium iodide double staining. The percentage of early apoptotic cells (annexin V-positive/propidium iodide-negative) is reported in the graph. (C) Ls174T cells were treated with PKF115-584 (0.5 μM), pyrvinium (50 nM) and FTS (100 μM), alone or in combination, for 24 hours and c-myc expression was evaluated by real-time PCR using GAPDH as a reference gene. Expression was normalized on vehicle-treated control cells. (D) The cells were incubated for 72 hours with inhibitors and cell lysates probed with anti-survivin and β-actin antibodies. (E–F) Ten thousand Ls174T cells were grown in soft-agar in the presence of PKF115-584 (0.1 μM), pyrvinium (25 nM), FTS (50 μM), alone or combined. Large colonies were counted 20 days later. The number of colonies formed by untreated control cells is set as 100% (E). Representative photographs are shown in panel (F). Statistical analysis: see legend to figure 2.

Figure 4. Expression analysis of selected genes after inhibitors treatments.

Ls174T cells were treated for 24 or 72 hours with inhibitors and a panel of genes focused on Wnt, Ras and apoptosis pathways was studied by QPCR using a 96-well array (A) or standard real-time PCR (B). The graphs in (B) show expression fold change compared to vehicle-treated controls. (C) Summary table reporting real-time PCR data analysis from all cell lines. Filled boxes indicate synergistic effect on gene expression (>2-fold change versus every single treatment). PY  =  pyrvinium.