Targeting focal adhesion turnover in invasive breast cancer cells by the purine derivative reversine

Abstract

Background: The dynamics of focal adhesion (FA) turnover is a key determinant for the regulation of cancer cell migration. Here we investigated FA turnover in a panel of breast cancer models with distinct invasive properties and evaluated the impact of reversine on this turnover in relation to cancer cell invasion in in vitro and in vivo conditions.

Methods: Live imaging and immunofluorescence assays were used to investigate FA turnover in breast cancer cells. Biochemical studies were used to investigate the impact of reversine on FA signalling and turnover. In vivo activity was investigated using orthotopic breast cancer mouse models.

Results: Accelerated FA disassembly from plasma membrane protrusions was observed in invasive compared with non-invasive breast cancer cells or non-immortalised mammary epithelial cells. Reversine significantly inhibited FA disassembly leading to stable FAs, which was associated with reduced cell motility and invasion. The inhibitory effect of reversine on FA turnover accounted for a large part on its capacity to interfere with FAK function on regulating its downstream targets. In orthotopic breast cancer mouse models, reversine revealed a potent inhibitory activity on tumour progression to metastasis.

Conclusion: These results support the utility of targeting FA turnover as a therapeutic approach for invasive breast cancer.

Figure 1

FA turnover in invasive breast cancer cells. (A) Quantification of focal adhesion disassembly (left panel) and assembly (right panel) in paired invasive and non-invasive breast cell lines. Measurements were made on at least 25 individual adhesions in 10 separate cells and presented as mean±s.d. (*P<0.05). (B–D) Effect of reversine on focal adhesion dynamics and structure in MDA231-M2 cells. Cells engineered to express GFP-paxillin were treated with or without 1 μM reversine. (B) Quantification of FA disassembly (left panel) and duration (right panel). Data presented as mean±s.d. from at least 10 cells per condition (*P<0.05 reversine vs DMSO). (C) Representative images of live cells obtained at the indicated time points. Arrowheads in the upper panel demonstrate dynamic turnover of focal adhesions, whereas arrows in the reversine-treated samples indicate stable focal adhesions, which are present throughout the study. (D) FA size (area) from MDA231-M2 cells treated with or without 1 μM reversine for 1 h were quantified, and representative averages were obtained from >40 FAs per group (*P<0.01 reversine vs DMSO (control)).

Figure 2

Effect of reversine on focal adhesion signalling components in MDA231-M2 cells. (A) Cells were treated with the indicated concentrations of reversine for 1 h. Protein lysates were then blotted and probed with anti-P-FAK Y397 and anti-FAK. The IC₅₀ for phospho-FAK inhibition was determined (right panel). (B) Cells were treated with 1 μM reversine or retreversine for 1 h. Protein was then blotted and probed with indicated antibodies. (C) Cellular FAK kinase activity was determined in MDA231-M2 cells treated with the indicated concentrations of reversine. FAK kinase assay was performed on immune-complexes from three independent experiments. (D) The effect of 1 μM reversine (Rv) or retreversine (Rt) on P-FAK Y397 was analysed in src-deficient (SYF^−/−) and proficient (SYF^+/+) cells. Phospho-FAK inhibition was observed in SYF^−/− cells.

Figure 3

Predicted binding mode of reversine in the ATP-binding pocket of FAK. Reversine is coloured by atomic-colouring scheme (C in green, N in blue and O in red). Residues within 3Å of the ligand are in cyan sticks. For clarity, only part of the FAK kinase domain is shown. Structure of reversine (right panel).

Figure 4

The effect of reversine (Rev) on the MDA231-M2 cell motility and invasion, as compared with controls (DMSO) and Retreversine (Ret). (A) Wound-healing assay of MDA231-M2 cells treated with 1 μM reversine, 1 μM Retreversine or DMSO. Arrows indicative of remaining wound area. (B–C) Cells were cultured in the upper chamber of the Boyden chamber in the absence or presence of reversine (1 μM; Rev), retreversine (1 μM; Ret) or DMSO. FBS was used as a chemoattractant in the lower chamber. (B) Representative photomicrograph of invading cells after 48 h incubation in the chamber, stained with haematoxylin. (C) Quantitiative representation of invading cells. Each bar represents the mean±s.d. (error bars) of invading cells of six independent experiments (*P<0.005 reversine vs DMSO, N=6). Cells treated with reversine demonstrated a significant reduction in their ability to invade.

Figure 5

Effect of reversine on tumour progression and tumour invasion in vivo as compared with control. (A) Primary tumour growth was monitored after subcutaneous implantation of MDA231-M2 or MDA468 cells into the flank of SCID mice and injection of control (saline) or reversine at 10 mg kg⁻¹ or 30 mg kg⁻¹. Tumour growth was monitored over time as indicated in Materials and Methods where the white bars represent the beginning of the treatment cycle and the black bars represent the day of sacrifice. Mice were treated bi-weekly for either 4 weeks (MDA231-M2) or 10 weeks (MDA-468). Each group represents the mean of eight mice±s.e.m. (*P<0.05). (B) Quantification of lung surface metastases of MDA231-M2 mice after taxol (7.5 mg kg⁻¹) or reversine (10 mg kg⁻¹ or 30 mg kg⁻¹) administration. Results are demonstrated as the percentage of inhibition of the mean number of lung metastases±s.e.m., as compared with control lungs (mean number of lung metastases in control lungs was equal to 335, n=8; *P<0.05).