GSK-3 inhibition overcomes chemoresistance in human breast cancer

Abstract

Glycogen Synthase Kinase-3β (GSK-3β), a serine/threonine protein kinase, is an emerging therapeutic target in the treatment of human breast cancer. In this study, we demonstrate that the pharmacological inhibition of GSK-3 by two novel small molecule GSK-3 inhibitors, 9-ING-41 and 9-ING-87, reduced the viability of breast cancer cells but had little effect on non-tumorigenic cell growth. Moreover, treatment with 9-ING-41 enhanced the antitumor effect of irinotecan (CPT-11) against breast cancer cells in vitro. We next established two patient-derived xenograft tumor models (BC-1 and BC-2) from metastatic pleural effusions obtained from patients with progressive, chemorefractory breast cancer and demonstrated that 9-ING-41 also potentiated the effect of the chemotherapeutic drug CPT-11 in vivo, leading to regression of established BC-1 and BC-2 tumors in mice. Our results suggest that the inhibition of GSK-3 is a promising therapeutic approach to overcome chemoresistance in human breast cancer, and identify the GSK-3 inhibitor 9-ING-41 as a candidate targeted agent for metastatic breast cancer therapy.

Keywords: 9-ING-41; Breast cancer; Chemoresistance; Drug development; GSK-3.

Conflict of interest statement

Conflict of interest

9-ING-41 has been licensed to Actuate Therapeutics, Inc. Alan Kozikowski, Andrey Ugolkov, Thomas O’Halloran and Andrew Mazar hold an equity interest in Actuate Therapeutics, Inc. Alan Kozikowski and Irina Gaisina are inventors on the 9-ING-41 patent.

Copyright © 2016. Published by Elsevier Ireland Ltd.

Figures

Fig. 1

Pharmacological inhibition of GSK-3 suppresses viability of breast cancer cells. (A) Whole cell lysate was prepared from breast cancer cells, separated by SDS-PAGE (50 μg/well), transferred to PVDF membrane, and immunoblotted as indicated. (B) Relative cell growth was measured by MTS assay in MDA-MB-468 and SKBR3 breast cancer cells treated with 9-ING-41 (9ING41) and 9-ING-87 (9ING87) for 72 hours as indicated. (C) Breast cancer cells were treated with GSK-3 inhibitors 9-ING-41 (9ING41) and 9-ING-87 (9ING87) for 36 hours and Western immunoblotting was performed as indicated. (D) Representative pictures of Hoechst staining of breast cancer cells treated with 9-ING-41 and 9-ING-87 as indicated for 48 hours. Fragmented apoptotic nuclei show intense Hoechst staining.

Fig. 2

Pharmacological inhibition of GSK-3 potentiates the effect of conventional chemotherapeutic drugs in breast cancer cells. (A) GSK-3 inhibitors 9-ING-87 and 9-ING-41 show lowest GI50 in growth inhibition of SKBR3 breast cancer cells as compared to other GSK-3 inhibitors AR-A014418, SB-216763 and LY2090314. (B) Relative cell growth was measured by MTS assay in breast malignant (SKBR3) and benign (MCF10A) cells treated with 9-ING-41 (0.5 μM), 9-ING-87 (1 μM) and AR-A014418 (20 μM) for 72 hours as indicated. (C) Relative cell growth was measured by MTS assay in breast cancer cell lines treated with 0.5 μM, 2 μM and 10 μM of 9-ING-41 for 3 hours and 72 hours as indicated. (D) Breast cancer cell lines were treated with 9-ING-41, CPT-11 or combination of 9-ING-41 with CPT-11 for 3 hours at indicated. After the treatment, drugs were replaced with fresh media and relative cell growth was measured by MTS assay after 72 hours. Columns, mean; bars, SE.

Fig. 3

Treatment with 9-ING-41 enhances the antitumor effect of CPT-11 in breast BC-1 PDX (ER+/PR+/HER2−) tumors. (A) Treatment history of breast cancer patient (case BC-1). Breast PDX tumor model was established after orthotopic injection of metastatic pleural effusion cells (obtained from breast cancer patient) in NSG mouse. Breast PDX tumor pieces were re-transplanted orthotopically to 20 mice (1 tumor per mouse). Tumors were size matched and mice were randomized into 4 treatment groups: control (DMSO; n = 4 mice), CPT-11 (5 mg/kg, n = 5 mice), 9-ING-41 (70 mg/kg, n = 5 mice) and CPT-11 + 9-ING-41 (n = 5 mice). (B) Vehicle or drugs were injected as indicated by arrows. Points, mean tumor volume; bars, SE. (C) Weight of resected tumors was measured. Columns, mean tumor weight; bars, SE. (D) Representative pictures of PDX subQ tumors from each group of animals. (E) Representative pictures of GSK-3β, phospho-Glycogen Synthase and β-catenin expression in control and 9-ING-41-treated BC-1 PDX tumors.

Fig. 4

Treatment with CPT-11 + 9-ING-41 leads to a regression of breast BC-2 PDX (ER+/PR+/HER2−) tumors. (A) Treatment history of breast cancer patient (case BC-2). Breast PDX tumor model was established after orthotopic injection of metastatic pleural effusion cells (obtained from breast cancer patient) in NSG mouse. Breast PDX tumor pieces were re-transplanted orthotopically to 16 mice (1 tumor per mouse). Tumors were size matched and mice were randomized into 4 treatment groups: control (DMSO; n = 4 mice), CPT-11 (20 mg/kg, n = 4 mice), 9-ING-41 (70 mg/kg, n = 4 mice) and CPT-11 + 9-ING-41 (n = 4 mice). (B) Vehicle or drugs were injected as indicated by arrows. Points, mean tumor volume; bars, SE. (C) Weight of resected tumors was measured. Columns, mean tumor weight; bars, SE. (D) Representative pictures of PDX subQ tumors from each group of animals. (E) Representative pictures of GSK-3β, phospho-Glycogen Synthase and β-catenin expression in control and 9-ING-41-treated BC-2 PDX tumors.