Src and CXCR4 are involved in the invasiveness of breast cancer cells with acquired resistance to lapatinib

Abstract

Lapatinib is a dual EGFR and ErbB-2 tyrosine kinase inhibitor that has significantly improved the clinical outcome of ErbB-2-overexpressing breast cancer patients. However, patients inexorably develop mechanisms of resistance that limit the efficacy of the drug. In order to identify potential targets for therapeutic intervention in lapatinib-resistant patients, we isolated, from ErbB-2-overexpressing SK-Br-3 breast cancer cells, the SK-Br-3 Lap-R-resistant subclone, which is able to routinely grow in 1 µM lapatinib. Resistant cells have a more aggressive phenotype compared with parental cells, as they show a higher ability to invade through a matrigel-coated membrane. Lapatinib-resistant cells have an increased Src kinase activity and persistent levels of activation of ERK1/2 and AKT compared with parental cells. Treatment with the Src inhibitor saracatinib in combination with lapatinib reduces AKT and ERK1/2 phosphorylation and restores the sensitivity of resistant cells to lapatinib. SK-Br-3 Lap-R cells also show levels of expression of CXCR4 that are higher compared with parental cells and are not affected by Src inhibition. Treatment with saracatinib or a specific CXCR4 antibody reduces the invasive ability of SK-Br-3 Lap-R cells, with the two drugs showing cooperative effects. Finally, blockade of Src signaling significantly increases TRAIL-induced cell death in SK-Br-3 Lap-R cells. Taken together, our results demonstrate that breast cancer cells with acquired resistance to lapatinib have a more aggressive phenotype compared with their parental counterpart, and that Src signaling and CXCR4 play an important role in this phenomenon, thus representing potential targets for therapeutic intervention in lapatinib-resistant breast cancer patients.

Keywords: CXCR4; ErbB-2; Src kinase; breast cancer; lapatinib; resistance; saracatinib.

Figure 1. Characterization of SK-Br-3 Lap-R cells. (A) Effects of lapatinib on the anchorage-dependent growth of SK-Br-3 and SK-Br-3 Lap-R cells. Cells were treated for 72 h with the indicated concentration of lapatinib, and cell proliferation was measured using an MTT assay. (B) Invasive ability of SK-Br-3 and SK-Br-3 Lap-R cells as determined by using a Boyden chamber-based colorimetric assay. Cells were allowed to invade for 20 h through a matrigel-coated membrane toward serum-free medium or 2% fetal bovine serum (FBS). OD: optical density. *P < 0.05, **P < 0.001 (Student t test).

Figure 2. Levels of activation of EGFR, ErbB-2 and ErbB-3 in parental and lapatinib-resistant cells. Phosphorylation of EGFR, ErbB-2, and ErbB-3 was assessed in SK-Br-3 cells in the absence or presence of lapatinib at a concentration of 140 nM, and in SK-Br-3 Lap-R cells untreated or treated with 140 nM or 1 µM lapatinib. Western blot analyses for phosphorylated or total EGFR, ErbB-2, and ErbB-3 were performed with specific antibodies.

Figure 3. Analysis of the activation of Src, ERK1/2 and AKT in SK-Br-3 and SK-Br-3 Lap-R cells. Western blot analysis for the expression of the activated forms of Src, AKT and ERK1/2 in parental SK-Br-3 cells treated with 140 nM lapatinib and/or 1 µM saracatinib and in SK-Br-3 Lap-R cells treated with 1 µM lapatinib and/or 1 µM saracatinib. The blot was normalized to α-tubulin.

Figure 4. Involvement of Src kinase in the proliferation and invasiveness of lapatinib-resistant cells. (A) Effects of treatment with saracatinib, alone or in combination with lapatinib, on the anchorage-dependent growth of SK-Br-3 and SK-Br-3 Lap-R cells. Cells were treated for 72 h with the indicated concentrations of the drugs and cell proliferation was determined using an MTT assay. (B) Combination analysis was performed using the method described by Chou and Talalay. CI, combination Index. (C) Effects of treatment with saracatinib on the invasive ability of SK-Br-3 and SK-Br-3 Lap-R cells, as determined by using a Boyden chamber-based colorimetric assay. OD, optical density. * P < 0.05, ** P < 0.001 (treated samples vs. control; Student t test).

Figure 5. Effects of CXCR4 inhibition on the invasive ability of SK-Br-3 Lap-R cells. (A) Western blot analysis for the expression of CXCR4 in SK-Br-3 and SK-Br-3 Lap-R cells in the absence or presence of the indicated concentration of lapatinib and/or saracatinib. α-tubulin was used for normalization. (B) Effects of different concentrations of the CXCR4 antibody on the invasive ability of SK-Br-3 and SK-Br-3 Lap-R cells. *P < 0.05, ** P < 0.001 for comparison between untreated vs. treated cells (Student t test). (C) Effects of the CXCR4 antibody (1 µg/ml) and the Src inhibitor saracatinib (0.5 µM), alone or in combination, on the invasiveness of SK-Br-3 Lap-R cells. OD, optical density. P < 0.05 when the combination was compared with (*) saracatinib- or (§) CXCR4 Ab-treated samples (Student t test).

Figure 6. Effects of treatment with TRAIL on survival in SK-Br-3 and SK-Br-3 Lap-R cells. Cells were treated with recombinant human TRAIL (60 ng/ml) and saracatinib (0.5 µM) for 5 h and apoptosis was assessed by analyzing Annexin V positive cells by flow cytometry. * P < 0.05, for comparison between untreated and treated cells (Student t test).