mTOR signaling mediates resistance to tankyrase inhibitors in Wnt-driven colorectal cancer

Activation of Wnt/β-catenin signaling is essential for colorectal carcinogenesis. Tankyrase, a member of the poly(ADP-ribose) polymerase (PARP) family, is a positive regulator of the Wnt/β-catenin signaling. Accordingly, tankyrase inhibitors are under preclinical development for colorectal cancer (CRC) therapy. However, Wnt-driven colorectal cancer cells are not equally sensitive to tankyrase inhibitors, and cellular factors that affect tankyrase inhibitor sensitivity remain elusive. Here, we established a tankyrase inhibitor-resistant cell line, 320-IWR, from Wnt/β-catenin-dependent CRC COLO-320DM cells. 320-IWR cells exhibited resistance to tankyrase inhibitors, IWR-1 and G007-LK, but remained sensitive to a PARP-1/2 inhibitor, olaparib, and several anti-CRC agents. In 320-IWR cells, nuclear localization of active β-catenin was decreased and expression of β-catenin target genes was constitutively repressed, suggesting that these cells repressed the Wnt/β-catenin signaling and were dependent on alternative proliferation pathways. 320-IWR cells exhibited upregulated mTOR signaling and were more sensitive to mTOR inhibition than the parental cells. Importantly, mTOR inhibition reversed resistance to tankyrase inhibitors and potentiated their anti-proliferative effects in 320-IWR cells as well as in CRC cell lines in which the mTOR pathway was intrinsically activated. These results indicate that mTOR signaling confers resistance to tankyrase inhibitors in CRC cells and suggest that the combination of tankyrase and mTOR inhibitors would be a useful therapeutic approach for a subset of CRCs.


MTT cell proliferation assay
For the measurement of cell viability, thiazolyl blue tetrazolium bromide (MTT) was added to the medium at a final concentration of 0.8 mg/ml and, after incubation for 4 h, the medium containing MTT was removed and dimethyl sulfoxide was then added. Optical density (OD) at 570 nm and 630 nm (reference) were measured using an xMark microplate spectrophotometer (Bio-RAD).

Cell cycle and apoptosis assay
Cell cycle change and apoptosis induction was determined by flow cytometry as described previously [35]. Briefly, after the treatment with tankyrase inhibitors, cells were fixed in 70% ethanol. After treatment with ribonuclease A, cells were stained with propidium iodide solution. Cell cycle distribution and apoptotic sub-G1 fraction of the cells were analyzed with a FACSCalibur flow cytometer (Becton Dickinson).

Sequencing of tankyrase genes
Total RNA was extracted and cDNA was synthesized as described in the RT-qPCR section of Materials and Methods. Protein coding regions of tankyrase-1 and -2 genes were amplified by PCR from the synthesized cDNA using the KOD Plus Neo kit (Toyobo Co., Ltd., Osaka, Japan) and were directly sequenced with 3130 Genetic Analyzer (Applied Biosystems).

TCF reporter assay
Reporter assay was done as described previously [30]. Cells were transfected with the firefly luciferase vector with Tcf promoter (pTcf7wt-luc, provided by Dr. Kunitada Shimotohno, National Center for Global Health and Medicine, via RIKEN BioResource Center, Ibaraki, Japan) and the Renilla luciferase vector phRL-CMV (Promega) by using Lipofectamine 2000 reagent (Thermo Fisher Scientific). Relative activity of firefly luciferase to Renilla luciferase was determined with the Dual-Glo Luciferase Assay System (Promega).

Cloning of 320-IWR cells
320-IWR cell-derived clones were obtained by limiting dilution method, seeding cells at the density of 1 cell/100 μL/well in 96-well plates. After two weeks, we obtained multiple single colonies from the plates, and expanded the clones for further analysis. #A of siRNAs to tankyrase-1 and -2 (siTNK #A) or cocktail #B of siRNAs to tankyrase-1 and -2 (siTNK #B) were prepared and introduced into the indicated cells as described in Supplementary Materials and Methods. (A) Forty-eight hours after siRNA introduction, cell lysates were prepared and protein levels of tankyrase and GAPDH (as a loading control) were evaluated by western blot analysis. For tankyrase detection, we used anti-tankyrase-1/2 antibody (sc-8337, Santa Cruz Biotechnology), which mainly detects tankyrase-1 in COLO-320DM cells (our unpublished analysis). (B) Forty-eight hours after siRNA introduction, total RNA was prepared and TNKS2 (tankyrase-2) expression levels were analyzed using RT-qPCR. β-Actin (ACTB) expression was analyzed to normalize the data. Error bars represent standard deviation (SD). (C) One-hundred twenty hours after siRNA introduction, cell number was evaluated as in Materials and Methods. Error bars represent standard deviation (SD) of three independent experiments. Statistical significance in the difference between COLO-320DM and 320-IWR cells was evaluated by Student t test (**: P < 0.01). RNA was prepared and the expression levels of the β-catenin target genes were analyzed using RT-qPCR. β-Actin (ACTB) expression was analyzed to normalize the data. Error bars represent standard deviation (SD). For MYC, LEF1 and JAG1 data, statistical evaluation was done by Student t test (**: P < 0.01). (B) COLO-320DM and 320-IWR cells were transiently transfected with the reporter plasmids, and the Tcf promoter activity normalized by CMV promoter activity (as a control) was determined. Error bars represent standard deviation (SD) of three independent experiments. Statistical evaluation was done by Student t test (**: P < .01).

Supplementary Figure 7: Synergistic anti-proliferative effect of tankyrase inhibitor and mTOR inhibitor in colorectal cancer cells. (A)
Effect of mTOR inhibitors temsirolimus and rapamycin on p70S6 kinase (p70S6K) in COLO-320DM and 320-IWR cells. Cells were treated with temsirolimus or rapamycin at the indicated concentrations for 2 h. Protein levels and phosphorylation status of p70S6K were evaluated by western blot analysis. (B) COLO-320DM and 320-IWR cells were treated with G007-LK and temsirolimus together at the indicated concentrations for 120 h. Cell numbers were evaluated as described in Materials and Methods. Error bars represent standard deviation (SD) of three independent experiments. Statistical significance was evaluated by Tukey-Kramer test (*: P < 0.05; **: P < 0.01). (C) Isobologram analysis was performed according to the calculation method as described [21]. In the graph, the combinational effect of two drugs is synergistic if the closed circle dot is left under the line. (D) HCC2998 and DLD-1 cells were treated with IWR-1 and temsirolimus at the indicated concentrations for 120 h. Cell numbers were determined as described in Materials and Methods. Error bars represent standard deviation (SD) of three independent experiments. Statistical significance was evaluated by Tukey-Kramer test (*: P < 0.05; **: P < 0.01). Phosphorylation level of mTOR pathway regulator, p70S6K in 320-IWR clones. Protein levels and phosphorylation status of p70S6K were evaluated by western blot analysis. (B) Reversal effect of temsirolimus on tankyrase inhibitor resistance of 320-IWR clones. Cells were treated with IWR-1 and temsirolimus (Temsiro) together at the indicated concentrations for 120 h. Cell numbers were calculated as described in Materials and Methods. Error bars represent standard deviation (SD) of three independent experiments. Statistical significance was evaluated by Tukey-Kramer test (*: P < 0.05; **: P < 0.01).