AZD3463, an IGF-1R inhibitor, suppresses breast cancer metastasis to bone via modulation of the PI3K-Akt pathway

Abstract

Background: The bone-derived insulin-like growth factor I (IGF-1) and its receptor IGF-1R play a crucial role in promoting the survival and proliferation of cancer cells, and have thus been considered as prime targets for the development of novel antitumor therapeutics.

Methods: By using the MDA-MB-231BO cell line, which is the osteotropic metastatic variant of the human breast adenocarcinoma cell line MDA-MB-231, and an in vivo model of breast cancer metastasis to bone, the current study evaluated the effect of AZD3463, an IGF-1R inhibitor, used alone or in combination with zoledronic acid (ZA), on the regulation of IGF-1R associated signal pathway and treatment of bone metastases (BM). Cell proliferation and invasion were measured by methyl thiazolyl tetrazolium (MTT) and Transwell assay respectively. Apoptotic cell number was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL).

Results: AZD3463 was shown to alleviate IGF-1R phosphorylation promoted by IGF-1 treatment in MDA-MB-231BO cells in a dose-dependent manner. In both the cells and the mouse model, 5 nM of AZD3463 stimulated cell apoptosis and suppressed proliferation on a level similar to that of 100 µM of ZA. Remarkably, the combined use of AZD3463 and ZA exhibited a synergistic effect and greater antitumor activity compared to when they were employed individually. Mechanistic investigations indicated that the apoptosis-inducing activity of AZD3463 could be associated to its role in the activation of the phosphoinositide 3-kinase (PI3K)-Akt signaling pathway.

Conclusions: These findings suggested that AZD3463 could serve as a promising therapeutic molecule for treating BM in breast cancer patients, particularly when applied in conjunction with ZA or other antitumor agents.

Keywords: Insulin-like growth factor I receptors (IGF-1R); bone metastases (BM); breast cancer; phosphoinositide 3-kinase-Akt pathway (PI3K-Akt pathway).

Figure 1

AZD3463 could attenuate IGF-1-promoted phosphorylation of IGF-1R in a dose-dependent manner. (A) Left: Western blot showing the levels of phosphorylated and total IGF-1R at various time points after IGF-1 exposure. Right: Western blot band intensities for Phospho-IGF-1R relative to β-actin were evaluated; (B) left: Western blot showing the levels of indicated proteins in the presence of a combination of 100 ng/mL of IGF-1 and varying concentrations of AZD3463. Right: Western blot band intensities for Phospho-IGF-1R relative to β-actin were evaluated. β-Actin was used as an internal control. The error bars represent the SD of the mean from three independent experiments. IGF-1, insulin-like growth factor I; IGF-1R, IGF-1 receptors.

Figure 2

AZD3463 could reduce the proliferation and migration of MDA-MB-231BO cells in a dose-dependent manner. (A) MTT assay showing the relative viable cell numbers in different experiment groups; (B) Transwell assay indicating a gradual decline in the number of migrated MDA-MB-231BO cells with increasing AZD3463 concentrations; (C) crystal violet staining images of migrating cells. Scale bar, 100 µm. *, P<0.05; **, P<0.01; ***, P<0.001. MTT, methyl thiazolyl tetrazolium; IGF-1, insulin-like growth factor I.

Figure 3

The combination of AZD3463 and ZA suppressed the proliferation and migration of MDA-MB-231BO cells. (A) MTT assay showing the relative viable cell numbers in different experiment groups; (B) Transwell assay indicating the numbers of migrated cells in different experiment groups; (C) crystal violet staining images of migrating cells. Scale bar, 100 µm. *, P<0.05; **, P<0.01. ZA, zoledronic acid; MTT, methyl thiazolyl tetrazolium; IGF-1, insulin-like growth factor I.

Figure 4

AZD3463 effectively promoted the apoptosis of MDA-MB-213BO cells through blocking the PI3K-Akt signaling pathway. (A) TUNEL assay denoting the percentage of cell apoptosis in different experiment groups (green, TUNEL-positive cells); (B) quantification of viable and apoptotic MDA-MB-213BO cells; (C) Western blot assay showing the levels of phosphorylated and total IGF-1R, phosphorylated and total Akt, as well as phosphorylated GSK-3β, Raf and PTEN in different experiment groups. β-actin was used as an internal control. Scale bar, 100 µm. PI3K-Akt, phosphoinositide 3-kinase; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling; IGF-1R, IGF-1 receptors; GSK-3β, glycogen synthase kinase 3 beta; ZA, zoledronic acid; IGF-1, insulin-like growth factor I.

Figure 5

The combination of AZD3463 and ZA inhibited breast cancer metastasis to bone in murine model. (A) Treatment strategies for different experiment groups; (B) respective radiologic analysis merged bioluminescent images and X-ray radiography; (C) BLI quantitation of tumor load in different experiment groups (n=6 for each group) 1 week and 5 weeks after the inoculation of MDA-MB-213BO cells; (D) respective images of IHC analyses of CK7 as a marker of breast cancer (top) and H&E (bottom) staining of bone tissue obtained from different experiment groups (n=6 for each group). The arrow indicated the invasive lesion in breast cancer. Scale bar, 100 µm. *, P<0.05; ***, P<0.001. ZA, zoledronic acid; BLI, bioluminescent imaging; H&E, hematoxylin-eosin; PBS, phosphate-buffered saline; IHC, immunohistochemistry.