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. 2019 Jan 15;144(2):389-401.
doi: 10.1002/ijc.31662. Epub 2018 Nov 29.

Activation of WNT/β-catenin Signaling Results in Resistance to a Dual PI3K/mTOR Inhibitor in Colorectal Cancer Cells Harboring PIK3CA Mutations

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Free PMC article

Activation of WNT/β-catenin Signaling Results in Resistance to a Dual PI3K/mTOR Inhibitor in Colorectal Cancer Cells Harboring PIK3CA Mutations

Ye-Lim Park et al. Int J Cancer. .
Free PMC article

Abstract

PIK3CA is a frequently mutated gene in cancer, including about ~15 to 20% of colorectal cancers (CRC). PIK3CA mutations lead to activation of the PI3K/AKT/mTOR signaling pathway, which plays pivotal roles in tumorigenesis. Here, we investigated the mechanism of resistance of PIK3CA-mutant CRC cell lines to gedatolisib, a dual PI3K/mTOR inhibitor. Out of a panel of 29 CRC cell lines, we identified 7 harboring one or more PIK3CA mutations; of these, 5 and 2 were found to be sensitive and resistant to gedatolisib, respectively. Both of the gedatolisib-resistant cell lines expressed high levels of active glycogen synthase kinase 3-beta (GSK3β) and harbored the same frameshift mutation (c.465_466insC; H155fs*) in TCF7, which encodes a positive transcriptional regulator of the WNT/β-catenin signaling pathway. Inhibition of GSK3β activity in gedatolisib-resistant cells by siRNA-mediated knockdown or treatment with a GSK3β-specific inhibitor effectively reduced the activity of molecules downstream of mTOR and also decreased signaling through the WNT/β-catenin pathway. Notably, GSK3β inhibition rendered the resistant cell lines sensitive to gedatolisib cytotoxicity, both in vitro and in a mouse xenograft model. Taken together, these data demonstrate that aberrant regulation of WNT/β-catenin signaling and active GSK3β induced by the TCF7 frameshift mutation cause resistance to the dual PI3K/mTOR inhibitor gedatolisib. Cotreatment with GSK3β inhibitors may be a strategy to overcome the resistance of PIK3CA- and TCF7-mutant CRC to PI3K/mTOR-targeted therapies.

Keywords: GSK3β; PI3K/mTOR dual inhibitor; TCF7 frameshift mutation; WNT/β-catenin signaling pathway; colorectal cancer; organoids; patient-derived colorectal cancer.

Figures

Figure 1
Figure 1
Gedatolisib sensitivity and resistance of CRC cell lines harboring PIK3CA mutations. (a) A total of 29 CRC cell lines were treated with vehicle or gedatolisib at the indicated concentrations for 72 h, and IC50 values were determined using the MTT viability assay. Untreated cells were considered 100% viable. (b) Western blot analysis of basal levels of the total or phosphorylated PI3K/mTOR pathway‐associated proteins in CRC cell lines. Data are representative of three independent experiments. β‐Tubulin was probed as a loading control in all western blots. (c) Colony‐forming assay of CRC cell lines treated with vehicle or gedatolisib at 0.1 or 1 μM for 10 days. (d) Annexin V‐FITC staining and western blotting of CRC cell lines treated with vehicle or gedatolisib at 1 μM for 72 h. (e) Human organoids, O28T(PIK3CA wild‐type) and O29T(PIK3CA R88Q), were treated gedatolisib at 0.01 or 0.1 or 1 μM for 6 days measured cell viability using the cell titler glo. (f) CRC cell lines were treated with vehicle or gedatolisib at 1 μM for 24 h, and cell lysates were subjected to western blotting with antibodies specific for total or phosphorylated proteins as indicated. [Color figure can be viewed at wileyonlinelibrary.com]
Figure 2
Figure 2
GSK3β sustains mTOR signaling in gedatolisib‐resistant cell lines. (a) HCT‐116 (sensitive) and HCT‐15 and LS174T (resistant) cells were treated with vehicle or gedatolisib at 1 μM for 12 h. TSC2 and Raptor were immunoprecipitated from cell lysates and subjected to western blotting with antibodies to TSC1 or TSC2 and mTOR or Raptor (upper rows). 30% of input cell lysate was blotted with antibodies to total or phosphorylated forms of the indicated proteins (lower rows). (b) Western blot analysis and phosphorylated and total GSK3β protein expression in the indicated CRC lines. The graph shows the ratio of p‐GSK3β(ser9)/p‐GSK3β(tyr216) to total GSK3β expression levels. (c) HCT‐15 and LS174T cells were transfected with control or GSK3β‐targeting siRNA for 48 h and then treated with vehicle or gedatolisib 1 μM for 12 h. mTOR was immunoprecipitated from cell lysates and subjected to western blotting with antibodies to Raptor or mTOR (Upper rows). 30% of input lysate was blotted with antibodies to total or phosphorylated forms of the indicated proteins. β‐Tubulin was probed as a loading control in all western blots. Data are representative of three independent experiments.
Figure 3
Figure 3
The WNT/β‐catenin pathway is hyperactivated in CRC cell lines rendered resistant to gedatolisib by the TCF7 H155 frameshift mutation. (a) Western blotting of basal levels of total and phosphorylated/active forms of the indicated WNT/β‐catenin pathway proteins. TOP/FOP flash luciferase assay was conducted in 7 cell lines under the Wnt3a stimulation. (b) Western blot analysis of total and phosphorylated/active forms of the indicated proteins in HCT‐15 and LS174T cells transfected with control or GSK3β‐targeting siRNAs and treated with vehicle or gedatolisib 1 μM for 24 h. (c) Viability of HCT‐116 and HT‐29 cells after transfection with Mock(empty vector) or TCF7 H155fs* (TCF7 MT) followed by treatment for 72 h with vehicle or 0.1 μM gedatolisib. The percentage of viable cells is shown relative to the untreated controls (*p < 0.05, ***p < 0.001). (d) HCT‐116 and HT‐29 parental and TCF7 MT cell lines were treated with vehicle or 0.1 μM gedatolisib for 24 h, and cell lysates were subjected to western blotting of total and phosphorylated/active forms of the indicated proteins. β‐Tubulin was probed as a loading control in all western blots. Data are representative of three independent experiments. (e) Viability of O29T organoids after transfection with Mock (empty vector) or TCF7 H155fs* (TCF7 MT) followed by treatment for 6 days with vehicle or 0.01 μM or 0.1 μM gedatolisib using cell tilter glo. The percentage of viable cells is shown relative to the untreated controls. [Color figure can be viewed at wileyonlinelibrary.com]
Figure 4
Figure 4
PI3K/mTOR and GSK3β inhibitors have synergistic effects on gedatolisib‐resistant CRC cell lines. (a) Upper: Colony‐forming assay in HCT‐15 and LS174T cells transfected with control or GSK3β‐targeting siRNAs and treated with vehicle or gedatolisib 1 μM for 10 days. Lower: Viability of HCT‐15 and LS174T cells transfected with control or GSK3β‐targeting siRNAs and treated with vehicle or gedatolisib 1 μM for 48 h. (***p < 0.001) (b) Annexin V‐FITC staining and western blotting of HCT‐15 and LS174T cells transfected with control or GSK3β‐targeting siRNAs and treated with vehicle or gedatolisib 1 μM for 48 h. (c) Western blot analysis of total and phosphorylated/active forms of the indicated proteins in HCT‐15 and LS174T cells transfected with control or GSK3β‐targeting siRNAs and treated with vehicle or gedatolisib 1 μM for 24 h. (d) Viability of HCT‐15 and LS174T cells at 72 h after treatment with the indicated combinations of vehicle, gedatolisib (1 μM), and/or the GSK3β inhibitor CHIR‐99021(10 μM and/or 20 μM) (***p < 0.001). (e) Colony‐forming assay of HCT‐15 and LS174T cells treated with the indicated concentrations of vehicle, gedatolisib, and/or CHIR for 10 days (***p < 0.001) (f) Annexin V‐FITC staining of HCT‐15 and LS174T cells treated with indicated combinations of vehicle, gedatolisib (1 μM), and/or the GSK3β inhibitor CHIR‐99021(10 μM and/or 20 μM) for 48 h. (g) Western blot analysis of basal levels of total and phosphorylated/active forms of the indicated proteins in HCT‐15 and LS174T cells. β‐Tubulin was probed as a loading control in all western blots. Data are representative of three independent experiments.
Figure 5
Figure 5
Co‐treatment with PI3K/mTOR and GSK3β inhibitors decreases CRC tumor growth in a xenograft mouse model. (a) Groups of 4‐week‐old female BALB/c nude mice were subcutaneously injected with HCT‐15 cells (1 × 107/mouse) and administered vehicle, gedatolisib (10 mg/kg), and/or LiCl (80 mg/kg) twice weekly for 3 weeks. Tumor size was measured every other day. Data are the mean ± s.d. of 5 mice per group. Representative pictures of HCT‐15 tumors excised from mice on day 19 of the experiment shown in (a). (b) Representative images of IHC staining of tumor tissues of mice with control and gedatolisib plus LiCl groups. H&E staining (upper) and Ki‐67 staining (lower). (c) Western blot analysis of the total and phosphorylated/active forms of the indicated proteins extracted from HCT‐15 tumors excised on day 19. β‐Tubulin was probed as a loading control.

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