Inhibition of the Axl pathway impairs breast and prostate cancer metastasis to the bones and bone remodeling

Abstract

Approximately 90% of cancer-related deaths result from cancer metastasis. In prostate and breast cancers, bone is the most common site of cancer cell dissemination. Key steps in the metastatic cascade are promoted through upregulation of critical cell signaling pathways in neoplastic cells. The present study assessed the role of the receptor tyrosine kinase Axl in prostate and breast cancer cell metastasis to bones using (i) Axl knockdown neoplastic cells and osteoclast progenitor cells in vitro, (ii) intracardiac injection of Axl knockdown tumor cells in vivo, and (iii) selective Axl inhibitor BGB324. Axl inhibition in neoplastic cells significantly decreased their metastatic potential, and suppression of Axl signaling in osteoclast precursor cells also reduced the formation of mature osteoclasts. In vivo, Axl knockdown in prostate and breast cancer cells significantly suppressed the formation and progression of bone metastases. Hence, therapeutic targeting of Axl may impair tumor metastasis to the bones through neoplastic and host cell signaling axes.

Keywords: Axl; Bone metastasis; Osteoclastogenesis; Receptor tyrosine kinase.

Fig. 1

Axl knockdown in breast and prostate cancer cells decreases migratory and invasive capacities. Axl was genetically inactivated by shRNA in MDA-MB-231 (a), PC3ML (b), and DU-145 (c) cell lines and two knockdown clones (shAXL#1 and shAXL#2) were selected by Western blot for further studies. Tumor cells were seeded in the transwell migration or invasion chambers. The number of migrated (d–e) or invaded (g–i) tumor cells were counted 24 h later. j Representative images of invasion chambers that were seeded with shSCM or shAXL DU-145 cells. Results are the mean and standard error values of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.0001, ****p < 0.00001

Fig. 2

A selective Axl inhibitor, BGB324, impairs tumor cell migration and invasion. MDA-MB-231 and PC3ML cells were seeded in the transwell migration or invasion chambers with varying concentrations of BGB324. The number of migrated (a, c) or invaded (b, d) tumor cells were counted 24 h later. Results are the mean and standard error values of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.0001, ****p < 0.00001

Fig. 3

Axl knockdown in MDA-MB-231 and PC3ML cells decreases metastasis to the bone. shSCM or shAXL MDA-MB-231 or PC3ML cells, expressing luciferase, were inoculated intracardiacally into 6–8 week-old female and male athymic nu/nu mice, respectively. Tumor seeding and progression was monitored via weekly bioluminescence imaging (BLI). Five weeks later, mice were euthanized. Representative bioluminescence images of mice inoculated with shSCM or shAXL MDA-MB-231 (a) and PC3ML (d) cells at the time of injection, week 3, and week 5. Total flux of intracardiacally injected shSCM and shAXL MDA-MB-231 (b) and PC3ML (e) cells were tracked over 5 weeks. Number of macroscopic metastases in mice inoculated with either shSCM or shAXL MDA-MB-231 (c) and PC3ML (f) cells at endpoint. MDA-MB-231: shSCM, n = 8; shAXL#1, n = 8; shAXL#2, n = 10; PC3ML: shSCM, n = 10; shAXL#1, n = 9; shAXL#2, n = 10. **p = 0.0036, ***p = 0.0006, ****p < 0.00001

Fig. 4

Pharmacologic inhibition of Axl inhibits osteoclast formation. Osteoclast progenitor cells were seeded in a 24-well plate (2 × 10⁴ cells/well), treated with or without 35 ng/ml RANK-L and varying concentrations of BGB324. Five days later, mature and differentiated osteoclasts were fixed and stained for tartrate resistant acid phosphatase (TRAP). a Representative images of TRAP-positive, multinucleated osteoclasts, 5X magnification. b Total number of TRAP-positive, multinucleated ($\ge$3 nuclei) osteoclasts, analyzed by One-way ANOVA. c Scheme of isolating primary osteoclast precursor cells from 6–8 week-old female BALB/c mice. d Representative images of TRAP-positive, multinucleated osteoclasts differentiated from primary osteoclast precursor cells of BALB/c mice, 5X magnification. e Total number of TRAP-positive, multinucleated ($\ge$3 nuclei) osteoclasts differentiated from primary osteoclast precursor cells, analyzed by Student’s t-test. Results are the mean and standard error values of three independent experiments. ***p < 0.0001, ****p < 0.00001

Fig. 5

Axl-expressing osteoclast precursor cells promote osteoclastogenesis. a Axl was genetically inactivated by shRNA in murine osteoclast progenitor cell line (Raw264.7), and two knockdown clones (shAXL#1 and shAXL#2) were selected by qPCR for further studies. shSCM and shAXL osteoclast progenitor cells were seeded in the transwell migration or invasion chambers. After 24 h, the number of migrated b Or invaded c Cells were counted. shSCM or shAXL osteoclast progenitor cells (2 × 10⁴ cells/well of a 24-well plate) were treated with 35 ng/ml RANK-L. Five days later, mature and differentiated osteoclasts were fixed and stained for tartrate resistant acid phosphatase (TRAP). d Representative images of TRAP-positive, multinucleated osteoclasts (indicated by the black arrows), 5X magnification. e Total number of TRAP-positive, multinucleated ($\ge$3 nuclei) osteoclasts were quantified. shSCM or shAXL osteoclast progenitor cells (2 × 10⁴ cells/well of a 24-well plate) were seeded on a dentine slice treated with or without 35 ng/ml RANK-L. Five days later, dentine slices were stained with toluidine blue solution to observe resorption pits on a dentine slice. f Representative images of bone slices from shSCM and shAXL cells treated with or without RANK-L, 10X magnification. g Total number of resorbed pits per bone slice were quantified.(*p < 0.05, **p < 0.01, ***p < 0.0001

Fig. 6

MCP-1 is a downstream effector of Axl in osteoclast precursor cells. Conditioned media of shSCM and shAXL osteoclast progenitor cells were collected and analyzed by cytokine array (a, b). c MCP-1 expression in shSCM and shAXL osteoclast progenitor cells were assessed by qPCR. Osteoclast progenitor cells were seeded and treated with or without 35 ng/ml RANK-L and/or 50 ng/ml MCP-1. Five days later, mature and differentiated osteoclasts were fixed and stained for tartrate resistant acid phosphatase (TRAP). d Representative images of TRAP-positive, multinucleated osteoclasts, 5X magnification. e Total number of TRAP-positive, multinucleated ($\ge$3 nuclei) osteoclasts were quantified. *p < 0.05, **p < 0.01, ***p < 0.0001