ALECOMM

Identification of drugs that block WNT-TCF pathway responses in human cancer cells mimicking the effects of dnTCF

We have used a transcriptional reporter assay for TCF activity driven by APC-insensitive N'∆β-CATENIN, to test a collection of clinical-trial tested small molecules (Microsource 1040 library) (Fig 1A). The optimized assay included a multimerized TCF-binding site firefly luciferase reporter in human 293T cells with a fivefold dynamic range as determined by maximal stimulation with active N'∆β-CATENIN and maximal repression with N'∆β-CATENIN plus dnTCF (see Varnat et al, 2010) (Fig 1A). All assays also included a Renilla luciferase control for normalization (see 4). Mutant TCF-binding site reporter controls for non-specific effects on general transcription were not part of the screen in order to streamline it, but this concern was directly addressed by testing the endogenous transcription of housekeeping genes by rt-qPCR (see below). The primary screen yielded a hit rate of 2.6% with > 55% inhibitory activity (Fig 1B). This excluded 3.3% of compounds considered cell-toxic as they repressed luciferase activity below that afforded by dnTCF.

 

The top nine candidates in the screen (Fig 1B, Supplementary Fig S1)—showing the highest repressed levels of normalized TCF-luficerase reporter activity above the baseline given by dnTCF—were then selected and tested for inhibition of human Ls174T colon cancer cell proliferation, as this is WNT-TCF-dependent (van de Wetering et al, 2002). BrdU incorporation analyses showed that only four out of the selected nine compounds repressed cell proliferation at > 50% as compared with DMSO-treated control cells (Fig 1C and D), yielding a secondary hit rate of ~ 0.4%.

Tertiary analyses measured the mRNA levels of an established cohort of canonical WNT-TCF target genes in Ls174T cells (van de Wetering et al, 2002; Hatzis et al, 2008) after 12 h of treatment to highlight early responses. Blockade of WNT-TCF by dnTCF4, used throughout our study as the genetic benchmark, yielded a pattern of gene expression that included both downregulated and upregulated genes, in comparison with control cells (Fig 1E). Expression levels were normalized by those of the ‘housekeeping’ genes TBP and HMBS (see 4). The expression of these genes was unaffected by the candidate compounds, providing an additional control against general transcriptional effects.

Of the 4 putative antagonists, only one, 4B5 (Avermectin B1), perfectly tracked the gene expression profile induced by dnTCF4 (Fig 1E, arrow). 4G11 (Oxybendazole) yielded a similar signature but failed to repress LGR5 to the same extend as 4B5 (Fig 1E). Although it harbors partial WNT-TCF antagonist activity, commercial Oxybendazole was largely inactive (B. Petcova, CM and ARA, data not shown). As in Ls174T cells, 4B5 treatment modified TCF targets and cell proliferation in primary human local colon cancer TNM4 CC14 cells (without detected mutations in APC or β-CATENIN) and in primary colon cancer liver metastasis mCC11 cells (Varnat et al, 2009) in a dose-dependent manner (Supplementary Fig S2). The final hit rate of the screen was ~ 0.1%.

Anti-proliferative activity of Abamectin, Ivermectin, Selamectin, and related macrocyclic lactones on human cancer cells

4B5 represents the anti-helmintic agent Avermectin B1, which belongs to the 16-membered Avermectin macrocyclic lactone family derived from Streptomyces avermitilis. It is a fermentation mixture of > 80% Avermectin B1a (5-O-demethyl-Avermectin A) and < 20% B1b (5-O-demethyl-25-de(1-methylpropyl)-25-(1-methylethyl-Avermectin A1) (S3). Ivermectin is a clinically approved Avermectin B1 derivative (> 90% 22,23-dihydroavermectinB1a and < 10% of 22,23-dihydroavermectinB1b) (Fig 2A, Supplementary Fig S3), used in humans against insect and worm infections, including river blindness caused by Onchocerca volvulus (Thylefors, 2008; Traore et al, 2012).

 

Ivermectin showed similar IC₅₀s on BrdU incorporation in vitro across several human colon cancer cells (IC₅₀: 1–2.4 μM) as Abamectin—the commercial name of Avermectin B1 (IC₅₀: 0.8–2 μM) (Fig 2B, Supplementary Fig S3). Likewise, Ivermectin and its commercial form from the pharmacy, Stromectol^™, showed similar inhibition of BrdU incorporation in vitro in two primary human glioblastomas and two human melanoma cell lines as compared with colon cancer cells (Fig 2B, Supplementary Fig S3).

Given that Avermectin B1 and Ivermectin are mixtures, we tested two commercial single-molecule Avermectin A1a derivatives, Doramectin, and Selamectin, to explore the activity of pure macrocyclic compounds. Doramectin (25-cyclohexyl-5-O-demethyl-25-de(1-methylpropyl) Avermectin A1a) and Selamectin (25-cyclohexyl- 4′-O-de(2,6-dideoxy-3-O-methyl-α-L-arabino-hexopyranosyl)- 5-demethoxy-25-de(1-methylpropyl)- 22,23-dihydro-5-(hydroxyimino)- Avermectin A1a) (Fig 2A and B, Supplementary Fig S3) are widely used in veterinarian medicine as anti-parasitics (Nolan & Lok, 2012). Doramectin was as effective as Ivermectin on reducing the proliferation of various human colon cancer cells (IC₅₀: 0.6–2.8 μM; Fig 2B, Supplementary Fig S3). In contrast, Selamectin, which scored as toxic in the primary screen at 10 μM, was ~ tenfold more potent, showing nanomolar BrdU incorporation IC₅₀s (0.08–0.14 μM, Fig 2B, Supplementary Fig S3). Tested in parallel, the Milbemycin macrocyclic lactone family member Moxidectin also displayed comparable anti-proliferative activity to Ivermectin (Fig 2B, Supplementary Fig S3). However, not all macrocyclic lactones showed such activity as Bryostatin, a distant macrocyclic lactone, was largely ineffective (90 and 88% BrdU incorporation vs. controls in DLD1 cells at 2.5 and 5 μM, respectively).

Ivermectin and Selamectin induce apoptosis

Ivermectin and Selamectin (Fig 2A) were chosen for further study given the EMEA- and FDA-approved status of the first and the high potency in vitro and widespread veterinarian use of the second. Activated Caspase3 was used as a marker of apoptosis by immunohistochemistry 48 h after drug treatment. Selamectin and Ivermectin induced up to a sevenfold increase in the number of activated Caspase3⁺ cells in two primary (CC14 and CC36) and two cell line (DLD1 and Ls174T) colon cancer cell types (Fig 2C). All changes were significative (P < 0.05) with the exception of 0.1 μM Selamectin in DLD1, CC14 and CC36, and were concentration and cell-type-dependent, with strongest effects detected in DLD1 cells with 2.5 μM Ivermectin (Fig 2C).

Ivermectin and Selamectin repress the expression of positive direct WNT-TCF targets

Gene expression analyses were performed 12 h after initiation of treatment to detect early responses. Importantly, both treatments repressed the direct positive WNT-TCF targets AXIN2, LGR5, and ASCL2 in DLD1 and Ls174T colon cancer cells, although they had variable effects on cMYC (Fig 2D). Minor differences in the levels of individual components of a WNT-TCF signature in different cells are expected (Herbst et al, 2014). In addition, both treatments also enhanced the levels of p21, a cell cycle blocker. This plus the finding that the expression of the ‘housekeeping’ genes TBP and HMBS used for normalization was unaffected, indicated the absence of general toxic effects on transcription.

Comparison of the effects of Ivermectin, Doramectin, Moxidectin, and Bryostatin on TCF targets in colon cancer cells

The inhibition of proliferation observed after treatment with different macrocyclic lactones (see above) begged the question of their relative abilities to block TCF responses in colon cancer cells. Analysis of the TCF targets AXIN2, LGR5, and p21 revealed equal potency of Ivermectin, Doramectin, and Moxidectin in Ls174T cells (Fig 2E). The distantly related macrocyclic lactone Bryostatin was inactive (Fig 2E). In primary CC14 cells, Ivermectin gave the strongest repression of AXIN2 and LGR5 and the strongest activation of p21, followed by Doramectin and by Moxidectin, all at the same concentration (Fig 2E). TCF target analyses (Fig 2D and E) thus confirmed the choice of Ivermectin and Selamectin for further studies.

Ivermectin and Selamectin inhibit clonogenic self-renewal of colon cancer stem cells

The strong downregulation of the expression of the intestinal stem cell genes ASCL2 and LGR5 (van der Flier et al, 2009; Schepers et al, 2012; Zhu et al, 2012b) by Ivermectin and Selamectin (Fig 2D) raised the possibility that these drugs could affect WNT-TCF-dependent colon cancer stem cell behavior. We thus tested for the ability of these drugs to antagonize cancer stem cell-driven clonogenic spheroid formation in vitro, which measures the ability of a cancer stem cell to self-renew and give rise to other progeny. Pretreatment of DLD1, CC14, and Ls174T attached cells in 2D culture with Ivermectin (1–2.5 μM) or Selamectin (0.01–0.1 μM) (Fig 3A) diminished the frequency of subsequent clonal floating spheroids by up to 73 and 43%, respectively, as compared with control, without affecting spheroid size (Fig 3B–E). Whereas this assay tests for an inhibitory effect of Ivermectin on the self-renewal of cancer stem cells in 2D culture that can then give rise to 3D spheroids, it does not directly address spheroid growth, that is self-renewal of the founding cancer stem cells or proliferation of derived non-stem cells in 3D culture.

 

To investigate the possibility that Ivermectin also affects proliferation in 3D spheroids as it does in 2D cultures, the drug was added at the beginning of clonal spheroid growth so that it would be present during spheroid formation. In this second test, Ivermectin decreased spheroid frequency in a dose-dependent manner (Supplementary Fig S4). Taken the results of the two distinct assays together, the data indicate that Ivermectin and Selamectin affect the frequency of clonogenic events in vitro as well as the growth of resulting clonal spheroids. These results suggest an action on both the bulk of the tumor and its cancer stem cells. Indeed, analyses of primary vs. secondary clonogenic events in Ls174T revealed that Ivermectin pre-treatment only affected primary cloning, without effects on secondary events far removed from the time of drug action (Fig 3F). This result also indicates the absence of long-lasting adverse drug effects.

Colon cancer cells with clonogenic spheroid capacity often express high levels of the CD133 (the AC133) epitope as compared with non-clonogenic cells (e.g., Varnat et al, 2009). Quantification of CC14 CD133⁺ cells by magnetic activated cell sorting after dissociation of 2D cultures with limited Trypsin or StemProAccutase treatments revealed no differences between control vs. (2.5 and 5 μM) Ivermectin-treated cells (7% vs 6.3%; P > 0.05). Ivermectin treatment thus separates CD133 expression and spheroid clonogenicity.

Ivermectin selectively represses TCF-dependent human colon cancer xenograft growth in vivo

Both genetic models of cancer and human xenografts provide highly valuable tools to model human disease (Richmond & Su, 2008). However, since we sought to test the effects of Ivermectin on human tumor cells, we chose to perform xenograft experiments.

There is general requirement of WNT-TCF signaling for human colon cancer cell proliferation in vitro (e.g., van de Wetering et al, 2002; Varnat et al, 2010), but this is not the case in vivo. We have shown that blocked WNT-TCF activity by expression of dnTCF4 in human colon cancer cells and primary xenografts in mice (including tumors generated by grafted Ls174T cells and primary CC14, CC36 cells) does not generally lead to arrested growth or cell death, with the exception of DLD1 cells (Varnat et al, 2010). We have thus used DLD1 vs. CC14 subcutaneous xenografts in Nude mice as a stringent differential test for the activity and specificity of Ivermectin in vivo. We reasoned that if this drug phenocopies genetic blockade of WNT-TCF responses by dnTCF in human epithelial cancer cells, it should block the growth of in-vivo-dnTCF-sensitive DLD1 tumors but not that of in-vivo-dnTCF-insensitive CC14 xenografts.

Intraperitoneal injections of cyclodextrin-conjugated Ivermectin given daily at 10 mg/kg after tumor establishment inhibited DLD1 tumor growth as compared with cyclodextrin carrier-only injected mice xenografted with sibling cells (Fig 4A). Critically, tumor inhibition by Ivermectin was equal to that observed with dnTCF4, our genetic benchmark (Fig 4A). Moreover, Ivermectin did not affect the growth of in-vivo-dnTCF4-insensitive CC14 tumors (Fig 4B). Injected mice showed no side effects and treatment with Ivermectin. Treatment with the commercial clinical-grade form of Ivermectin, Stromectol^™, also had positive effects (Supplementary Fig S5). The inhibition of DLD1 tumors, however, was size-dependent as starting the treatment when subcutaneous xenografts were large (> 100 mm³) yielded a poor response (8/9) to Ivermectin treatment via IP (not shown), possibly due to ineffective drug penetration.

 

To extend in vivo data to a second WNT-dependent colon cancer type, we used HT29, which harbors a similar truncation in APC as DLD1 cells. Subcutaneous injection of HT29 cells into the flanks of Nude mice yielded tumors, the growth of which was repressed by Ivermectin treatment (Fig 4C). Moreover, the overall pattern of TCF target gene regulation in HT29 cells in response to Ivermectin treatment was overall similar to that detected in DLD1 cells (Fig 2D and 4D).

Ivermectin inhibits lung cancer xenograft growth in vivo

Beyond colon cancer, WNT-TCF signaling has been implicated in a number of other tumor types including advanced non-small cell lung cancer (Nguyen et al, 2009; Pacheco-Pinedo et al, 2011). We have therefore used H358 human metastatic lung bronchioalveolar carcinoma cells to test for a response to Ivermectin at identical doses as for colon cancer cells. Pre-established H358 tumors responded to Ivermectin showing a ~ 50% repression of growth (Supplementary Fig S5). Consistently, Ivermectin treatment repressed a lung cancer WNT-TCF signature that included the direct targets AXIN2, LEF1, SOX4, and CYCLIND1 (as ASCL2 and LGR5 are not expressed in these cells) and enhanced p21 levels (Supplementary Fig S5). Ivermectin also diminished the protein levels of CYCLIN D1, a direct TCF target and oncogene, in both HT29 and H358 tumor cells (Fig 4D, Supplementary Fig S5).

Direct activation of TCF rescues the effects of low doses of Ivermectin and Selamectin

To further investigate the specificity of Ivermectin and Selamectin for the WNT-TCF pathway, we attempted to rescue their effects in colon cancer cells by directly enhancing TCF function via TCF^VP16, a fusion of the TCF DNA-binding domain plus the strong VP16 viral transcriptional transactivator (Kim et al, 2000) that acts in a WNT- and β-CATENIN-independent manner (Supplementary Fig S6). TCF^VP16-expressing cells were insensitive to low concentrations of Ivermectin or Selamectin in vitro in BrdU incorporation assays, with full rescue observed at 0.5–1 μM for Ivermectin and at 0.05–0.1 μM for Selamectin as compared with controls in DLD1, Ls174T, and primary TNM3 CC36 cells (8) (Fig 5A, Supplementary Fig S6). At higher concentrations, these drugs likely engage additional mechanisms (see 3).

 

TCF^VP16 expression led to strongly boosted positive WNT-TCF target gene levels in colon cancer cells, shown as ratios of those in Ivermectin-treated TCF^VP16 cells over those in Ivermectin-treated control cells (Fig 5B). Low concentrations of Ivermectin (1 μM) or Selamectin (0.1 μM) also repressed the normal levels of CYCLIN D1 protein by 90 and 50%, respectively, detected in DMSO-treated controls, and this repression was fully reversed by expression of TCF^VP16 (Fig 5C).

Importantly, sustained expression of TCF^VP16 through integrated lentivectors in DLD1 cells rescued the growth blockade of pre-established xenografts by Ivermectin (Fig 5D).

Ivermectin and Selamectin decrease the levels of C-terminal phosphoforms of β-CATENIN and the levels of the direct TCF target CYCLIN D1

Since Ivermectin repressed TCF reporter activity driven by APC-insensitive N'∆β-CATENIN, we sought to test whether this drug could affect downstream activation of β-CATENIN/TCF, focusing on C-terminal phosphorylation of β-CATENIN itself, which is required for full TCF complex transcriptional activity (Hino et al, 2005; Fang et al, 2007; Zhao et al, 2010). Treatment of DLD1 and Ls174T colon cancer cells in vitro with Ivermectin led to a dose-dependent inhibition of the levels of two C-terminal phosphoforms of β-CATENIN: Phospho-Ser552 and Phospho-Ser675 and to the repression of CYCLIN D1 (Fig 6A, Supplementary Fig S7). GAPDH and total β-CATENIN levels were unchanged (Fig 6A, Supplementary Fig S7).

 

The inhibitory activity of Ivermectin and Selamectin on CYCLIN D1 levels requires the function of serine protein phosphatases

Decreased levels of β-CATENIN phosphoforms could, in principle, result from the inhibition of kinases that phosphorylate C-terminal sites or from the superactivation of phosphatases that dephosphorylate these sites. Therefore, to test for an effect of Ivermectin in promoting the loss of C-terminal β-CATENIN phosphorylation, we first assayed for a possible rescue by okadaic acid (OA), which blocks the activity of the serine protein phosphatases PP2A and PP1. As expected, treatment with OA led to elevated levels of β-CATENIN C-terminal phosphoforms and of the TCF target CYCLIN D1 (Supplementary Fig S8). However, Ivermectin was ineffective in reducing OA-enhanced expression (Fig 6C and D, Supplementary Fig S8) as in controls (Fig 6A,C and D). Similar results for CYCLIN D1 were also obtained in lung cancer H358 cells (Fig 6B). These results suggest that Ivermectin requires active PP2A/PP1 to exert its repressive effect on the WNT-TCF pathway.

To complement these findings, we sought to superactivate the WNT-TCF pathway by enhancing kinase activity through the activation of endogenous Protein Kinase A, which phosphorylates β-CATENIN at C-terminal sites (Hino et al, 2005), with forskolin (FK). Treatment with FK was unable to rescue the inhibitory effect of Ivermectin on the levels of C-terminal phosphoforms of β-CATENIN and of CYCLIN D1 (Fig 6C and D), indicating that, unlike hyperphosphorylation by phosphatase inhibition, hyperphosphorylation via enhanced kinase activation is subject to repression by Ivermectin.

Finally, these effects were fully reproduced by Selamectin, where OA but not FK, rescued its effects (Fig 6E and F), suggesting a similar mode of action for these two macrocyclic lactones on WNT-TCF signaling (Fig 6H).

Localization of β-CATENIN after Ivermectin treatment

As a further test for the action of Ivermectin on β-CATENIN, we mapped its subcellular distribution by immunocytochemistry. Analyses of multiple samples showed similar distributions in control (DMSO) and Ivermectin (5 μM)-treated CC14 cells 6 h after treatment (Fig 6G). In both cases membrane, cytoplasmic and nuclear signals were detected.

Cell culture

Human colon cancer CC14 and CC36 primary cells and Ls174T and HT29 cell lines were cultured as previously described (Varnat et al, 2009, 2010). Colon cancer DLD1 and lung non-small cell bronchioalveolar carcinoma H358 cells were cultured as attached 2D layers in DMEM-F12 or RPMI 1640 with 10% FBS. 293T cells were cultured in DMEM with 10% FBS. For drug treatments, cells were plated in 2% FBS and treated the next day for 12–48 h. GBM primary cells were cultured as described in Zbinden et al (2010). U251 glioma and SKMel2 melanoma cell lines were cultured as described by ATCC.

Primary screening

A luciferase reporter assay was performed in 293T cells to identify compounds able to decrease TCF-driven gene expression using a TCF-binding site fused to a firefly luciferase reporter construct. Activation was driven by transfected N'∆β-CATENIN acting on endogenous TCF factors. Internal controls derived from Renilla luciferase activity resulting from a HSV-TK expression plasmid co-transfected in all cases. Transfection of 293T cells was preformed with calcium phosphate and plated at a density of 5,000/well into 96-well plates. Each transfection combined a constitutively active β-CATENIN plasmid as effector, a TCF-binding site firefly luciferase plasmid as reporter, and a HSV-TK Renilla luciferase plasmid as control. Cells were harvested 12 h after compounds treatment. Secondary reporter assays were repeated at least three independent times.

Drugs

Abamectin (31732, Fluka), Ivermectin (Fagron Iberica, and Sigma #I8898), Doramectin (33993, Fluka), Selamectin (32476, Sigma), Stromectol^™ (Merck) and Moxidectin (33746, Sigma) were diluted in 100% DMSO or ethanol and used at 0.01–10 μM for in vitro assays. Okadaic acid (OA, Enzo Life Science) was used at 1–15 nM.

BrdU incorporation

Colon primary cell culture and cell lines were plated in medium containing 2% FBS and treated with different drug concentrations for 48 h. After a 15′ pulse with BrdU (10 mg/ml), cells were fixed in 4% PFA, incubated in 2N HCl for 15′, neutralized with 0.1 M Boric acid for 10′, blocked in PBS-10% HINGS for 10′, and labeled with anti-BrdU antibodies (1:5,000, University of Iowa Hybridoma Bank). Signal was revealed with a rhodamine-coupled anti-mouse secondary antibody (1:500, Invitrogen Molecular Probes) and counterstained with DAPI (Sigma). At least 10 fields were quantified for each condition.

Apoptosis assay

Colon cancer cells were plated at 30% confluency and treated with different drug concentrations for 48 h. After treatment, cells were fixed in 4% PFA for 2 min and blocked in 10% HINGS for 1 h. Apoptosis was evaluated by immunofluorescence using a rabbit anti-cleaved Caspase3 antibody (1:200, overnight at 4°C; Cell Signaling) and Cy3-labeled secondary antibody (1:500, 1 h at RT; Jackson ImmunoResearch). Cells were counterstained with DAPI (1:10,000, Sigma). At least 10 fields were quantified per plate for each condition.

Quantitative reverse transcription PCR

RNA extraction, reverse transcription and qPCR were performed as previously described (Varnat et al, 2009).

qPCR primer sequences were as follows (5′->3′):

In vitro clonogenic assays and CD133 sorting

Adherent colon cancer cells were treated with Ivermectin or Selamectin at different concentrations as described, washed, lifted, and plated at 1 cell/well in 96-well non-adherent plates in colon spheroid medium (DMEM-F12 supplemented with B27 and 10 ng/ml EGF) without drugs and grown for 14 days. Spheres were then counted visually using an inverted Zeiss optical microscope. For secondary clonogenic assays, primary spheres were dissociated and replated at 1 cell/well in 96-well plates and cultured as described above. Each experiment was repeated at least thrice. Alternatively, untreated cells were plated and treated in 96-well plates with drugs and subsequently scored for spheroid formation. CD133 MACS (Miltenyi Biotech) was performed as described previously (Varnat et al, 2009, 2010) using either limited Trypsin or StemProAccutase (Gibco LifeTech) for cell dissociation.

Western blots

Cells were plated in media containing 2% FBS and treated with Ivermectin or Selamectin at different concentrations for 12 h. Protein lysates were in RIPA buffer. 10–20 μg of protein were loaded onto 12% polyacrylamide gels and the proteins transferred onto nitrocellulose membranes (Hybond), which were probed with primary and secondary antibodies as noted. Signal was revealed using the ECL system (GE Healthcare) and film. ImageJ was used for quantification. Membranes were sequentially reprobed after washing in TBS-1% Tween-20 and controlling for loss of signal. The following primary antibodies were used: anti-GAPDH (1:6,000 for 1 h at RT, 2118 Cell Signaling), anti-cyclinD1 (1:100 for 1 h at RT, Ab-3 Oncogene research), anti-Pser552 β-CATENIN (1:1,000 for 1 h at RT, 9566 Cell Signaling), anti-Pser675 β-CATENIN (1:1,000 for 1 h at RT, 4176 Cell Signaling), and anti-total β-CATENIN (1:1,000 for 1 h at RT, 8480 Cell Signaling). As secondary antibodies, HRP-coupled anti-rabbit or anti-mouse (1:6,000, Promega) were incubated for 1 h at RT.

Mouse xenografts

5 × 10⁵ control or lentivector-infected cancer cells were injected subcutaneously into the flanks of 6- to 8-week-old female NMRI Nude mice. After tumor establishment, mice were treated with cyclodextrin carrier alone or Ivermectin (or Stromectol^™) conjugated with cyclodextrin (45%) via daily intraperitoneal injections at 10 mg/kg. Tumor volumes were measured every 2–3 days.

Sequencing APC and β-CATENIN commonly mutated regions

One-hundred nanograms of genomic DNA from primary colon cancer cells (CC14 and CC36) or cell lines (DLD1 and Ls174T) was used for PCR using Phusion HF DNA Polymerase (BioLabs). Specific primers were designed to amplify commonly mutated regions in APC and CTNNB1 (β-CATENIN) genes. The Mutation Cluster Region (MCR) of APC was amplified with two pairs of primers: (1) MCR1FWD 5′-GATACTCCAATATGTTTTTC-3′ and MCR1REV 5′-GGAAGATCACTGGGGCTTAT-3′, (2) MCR2FWD 5′-GTGAACCATGCAGTGGAATG-3′ and MCR2REV 5′-TCTGAATCATCTAATAGGTC-3′. The primers for CTNNB1 were as follows: CTNNB1FWD 5′-CAATGGGTCATATCACAGATTCTT-3′ and CTNNB1REV 5′-TCTCTTTTCTTCACCACAACATTT-3′.

β-CATENIN immunohistochemistry

Colon cancer cells were plated at 40% confluency and treated with 5 μM Ivermectin or 2% DMSO for 6 h. After treatment, cells were fixed in fresh 4% PFA for 2 min. Antigen retrieval was conducted by boiling for 7 min in 10 mM citrate buffer (10 mM citric acid, 0.05% Tween-20, pH 6.0). Cells were then incubated at RT for 30 min, washed with PBS and permeabilized in PBS with 1% Tween-20 for 15 min at RT. Anti-β-CATENIN antibodies (1:400, Cell Signaling) were applied overnight at 4°C after blocking with 3% BSA in PBS. Signal was revealed with a rhodamine-coupled anti-rabbit secondary antibody (1:1,000) and counterstained with DAPI (1:10,000). Samples were imaged with Zeiss LSM 510META confocal microscope. Five independent slides were analyzed per each condition.