P3-17-04: CXCR1/2 Regulates Human Breast Cancer Stem Cell Activity Via EGFR/HER2−Dependent and -Independent Pathway.
Authors: JK Singh, G Farnie, RB Clarke, NJ Bundred
Journal: Cancer Research (2011)

Abstract Background Increasing evidence suggests that breast cancers are sustained by breast cancer stem cells (CSCs). Interleukin-8 (IL-8) via its cognate receptors, CXCR1 and CXCR2, and HER2 regulate CSC activity. Cytokine receptors can trans-activate members of the epidermal growth factor receptor (EGFR) family in normal and malignant tissue. The aims of this study were to determine (1) the effect of the IL-8-CXCR1/2 signalling pathway on human breast CSC activity, (2) the effect of CXCR1/2 inhibition in combination with EGFR/HER-2 inhibition on CSC activity and (3) the interaction of CXCR1/2 and EGFR/HER2 in breast cancer. Methods: Human breast cancer cell lines used were HER2−18 and SKBR3 (both HER2 over-expressing). Primary human breast cancer cells were extracted from metastatic samples (pleural effusion n=4; ascites n=5; local recurrence n=1) and invasive breast cancers (n=1). CSC activity was investigated in vitro using the mammosphere assay. Mammosphere forming efficiency (MFE) was determined by dividing the number of mammospheres &amp;gt;60μm in diameter divided by the number of cells seeded and expressed as a percentage. Data is represented as mean MFE ± standard error of mean normalised to control. Statistical significance (P) was determined using Mann-Whitney U test (2-tailed). Inhibitors used were SCH563705 (a CXCR1/2 inhibitor; 100nM), Lapatinib (an EGFR/HER-2 inhibitor; 1–10μM) and Gefitinib (an EGFR inhibitor; 1μM). Results: IL-8 (100ng/ml) increased HER2−18 mean MFE from 1±0.05% to 1.20±0.04%; 19.5% increase; P=0.0061. This effect was reduced by SCH563705 (from 1.20±0.04% to 0.70±0.05; 41.7% reduction; P=0.0001) and Lapatinib (from 1.20±0.04% to 0.51±0.04; 57.5% reduction; P &amp;lt;0.0001). Combination treatment resulted in a greater reduction in mean MFE compared to either SCH563705 (P&amp;lt;0.0001) or Lapatinib (P=0.026). IL-8 (100ng/ml) increased mean MFE of primary human breast cancer cells from 1±0.03% to 1.43±0.06%; 42.9% increase; P&amp;lt;0.0001 (n=11 patient samples). This effect was reduced by SCH563705 from 1.43±0.06% to 0.96±0.04%; 32.9% reduction; P&amp;lt;0.0001. In HER2 over-expressing patient samples (n=4), IL-8-induced increase in MFE was reduced by SCH563705 (from 1.43±0.06% to 0.83±0.05; 42.0% reduction; P&amp;lt;0.0001) and Lapatinib (from 1.43±0.06% to 0.91±0.05%; 36.4% reduction; P&amp;lt;0.0001). Combination treatment resulted in a greater reduction in mean MFE compared to either SCH563705 (P=0.0019) or Lapatinib (P&amp;lt;0.0001). In HER2−non-over-expressing samples (n=6), IL-8-induced increase in MFE was reduced by SCH563705 (from 1.45±0.10% to 1.06±0.05; 27.0% decrease; P=0.0005) and Lapatinib (from 1.45±0.10% to 1.15±0.07%; 20.7% decrease; P=0.0145). Combination treatment had no additional effect. Western blot analysis of the HER2−18 and SKBR3 cell lines showed that IL-8 induced phosphorylation of HER-2, AKT and MAPK within 10 minutes; these effects were inhibited by Lapatinib and Gefitinib. Conclusions: IL-8/CXCR1/2 regulates the activity of human breast CSCs. Lapatinib inhibited IL-8 signalling suggesting that CXCR1/2 signal transduction involves an EGFR/HER2−dependent pathway. Targeting CXCR1/2 adds to the efficacy of targeting HER2 in HER2−over-expressing human breast CSCs through inhibition of an EGFR/HER2−independent pathway. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-17-04.