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. 2017 Jul 18;8(29):47902-47915.
doi: 10.18632/oncotarget.18146.

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

Tetsuo Mashima  1 , Yoko Taneda  1   2 , Myung-Kyu Jang  1   2 , Anna Mizutani  1 , Yukiko Muramatsu  1 , Haruka Yoshida  1 , Ayana Sato  1   2 , Noritaka Tanaka  1   3 , Yoshikazu Sugimoto  3 , Hiroyuki Seimiya  1   2
Affiliations

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

Tetsuo Mashima et al. Oncotarget. .

Abstract

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.

Keywords: Wnt; colorectal cancer; mTOR; resistance; tankyrase.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Tankyrase inhibitors suppress colorectal cancer COLO-320DM cell proliferation through inhibition of the β-catenin pathway
(A) Expression of constitutively active β-catenin in COLO-320DM cells. Cells were transfected with the plasmid vector expressing active β-catenin (Ser45Δ) with a FLAG epitope tag or control vector (Mock) as described in the Materials and Methods. At 24 h after transfection, cells were left untreated or treated with IWR-1 for 16 h. Protein levels of the exogenously expressed constitutively active β-catenin (FLAG), active β-catenin which was dephosphorylated on Ser37 or Thr41 (endogenous and exogenous) and Axin1 and 2 were evaluated by western blot analysis. (B) Effect of constitutively active β-catenin expression on tankyrase inhibitor-induced growth inhibition of COLO-320DM cells. COLO-320DM cells transfected with active β-catenin (Ser45Δ) or control vector (Mock) were treated with tankyrase inhibitors, IWR-1 or G007-LK, for 120 h. Cell numbers were evaluated as described in the 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).
Figure 2
Figure 2. Establishment of 320-IWR, a tankyrase inhibitor-resistant sub-cell line of COLO-320DM cells
(A, B) Selective resistance of 320-IWR cells to tankyrase inhibitors. COLO-320DM and 320-IWR cells were treated with IWR-1 or G007-LK (A) or with olaparib, regorafenib, 5-fluorouracil (5-FU), or SN38, the active metabolite of irinotecan (B) for 120 h. Cell numbers were evaluated as 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) Effect of tankyrase inhibitors on tankyrase protein levels in COLO-320DM and 320-IWR cells. Cells were treated with IWR-1 or G007-LK at the indicated concentrations for 16 h. Protein levels of tankyrase and GAPDH as a loading control were evaluated by western blot analysis.
Figure 3
Figure 3. Alteration of canonical Wnt/β-catenin signaling in 320-IWR cells
(A, B) Effect of tankyrase inhibitors on protein levels of Wnt/β-catenin pathway regulators in COLO-320DM and 320-IWR cells. Cells were treated with IWR-1 and G007-LK at the indicated concentrations for 16 h. Protein levels of Axin1, 2, active β-catenin (dephosphorylated on Ser37 or Thr41) and GAPDH as a loading control were evaluated by western blot analysis.
Figure 4
Figure 4. Constitutively repressed Wnt/β-catenin signaling in 320-IWR cells
(A) Subcellular localization of β-catenin in COLO-320DM and 320-IWR cells. Cells were left untreated (upper panels and control in lower panels) or treated with 3 μM IWR-1 or 3 μM G007-LK for 16 h (lower panels). Cells were subjected to immunofluorescence staining with anti-non-phospho-β-catenin (green). DAPI staining of nuclear DNA is shown in white. (B) Expression of genes downstream of the Wnt/β-catenin pathway in COLO-320DM and 320-IWR cells. Cells were left untreated (−) or treated with 3 μM IWR-1 for 16 h. Total RNA was prepared and the expression levels of the Wnt/β-catenin pathway genes were analyzed using RT-qPCR. β-Actin (ACTB) expression was analyzed to normalize the data. Error bars represent standard deviation (SD) of three independent experiments. Statistical significance between untreated and IWR-1-treated COLO-320DM cells or between untreated COLO-320DM and 320-IWR cells was evaluated by Student t test (*: P < 0.05; **: P < 0.01).
Figure 5
Figure 5. Activation of mTOR signaling pathway in tankyrase inhibitor-resistant 320-IWR cells
(A) Representative result of the Gene Set Enrichment Analysis (GSEA) showing enrichment of the AKT–mTOR pathway-related gene signature in 320-IWR cells. (B) Elevated phosphorylation of mTOR pathway regulators in 320-IWR cells. Cells were left untreated or treated with 3 μM IWR-1 for 16 h. Protein levels and phosphorylation status of mTOR pathway regulators were evaluated by western blot analysis. (C) Preferential sensitivity of 320-IWR cells to mTOR inhibitors. COLO-320DM and 320-IWR cells were treated with mTOR inhibitors, temsirolimus or rapamycin, for 120 h. Cell numbers were evaluated as in the 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).
Figure 6
Figure 6. mTOR pathway activation confers resistance to tankyrase inhibition in 320-IWR cells
(A) Inhibition of p70S6 kinase (p70S6K) phosphorylation by an mTOR inhibitor. Cells were treated with temsirolimus (Temsiro) at the indicated concentrations for 2 h. Protein levels and phosphorylation status of p70S6K were evaluated by western blot analysis. (B) Reversal of tankyrase inhibitor resistance in 320-IWR cells by temsirolimus. COLO-320DM and 320-IWR cells were treated with IWR-1 and 4 nM temsirolimus (Temsiro) together at the indicated concentrations for 120 h. Cell numbers were calculated as in the 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) Expression and phosphorylation of p70S6 kinase (p70S6K) and 4E-BP1 in CRC cells. Protein levels and phosphorylation of p70S6K were evaluated by western blot analysis. (D) Potentiation of the anti-proliferative effect of IWR-1 by temsirolimus. Cells were treated with IWR-1 at the indicated concentrations and 10 nM temsirolimus (Temsiro) together for 120 h. Cell numbers were calculated. Error bars represent standard deviation (SD) of three independent experiments. Statistical significance was evaluated by Tukey-Kramer test (**: P < 0.01).
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