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. 2008 Mar 25;98(6):1076-84.
doi: 10.1038/sj.bjc.6604278. Epub 2008 Mar 11.

Activity of lapatinib a novel HER2 and EGFR dual kinase inhibitor in human endometrial cancer cells

G E Konecny  1 , N Venkatesan, G Yang, J Dering, C Ginther, R Finn, M Rahmeh, M Schoenberg Fejzo, D Toft, S-W Jiang, D J Slamon, K C Podratz
Affiliations

Activity of lapatinib a novel HER2 and EGFR dual kinase inhibitor in human endometrial cancer cells

G E Konecny et al. Br J Cancer. .

Abstract

In this study, we explore the therapeutic potential of lapatinib a selective inhibitor of both the EGFR and HER2 tyrosine kinases for the treatment of endometrial cancer. The effect of lapatinib on tumour cell growth and receptor activation was studied in a panel of human endometrial cancer cell lines. Candidate molecular markers predicting sensitivity were assessed by baseline gene expression profiling, ELISA, and western blot analyses. Multiple drug effect/combination index (CI) isobologram analysis was used to study the interactions between chemotherapeutic drugs and lapatinib. Concentration-dependent anti-proliferative effects of lapatinib were seen in all endometrial cancer cell lines tested, but varied significantly between individual cell lines (IC(50) range: 0.052-10.9 micromol). HER2 overexpression or increased expression of EGFR was significantly associated with in vitro sensitivity (P=0.024 or 0.011, respectively). Lapatinib exerts growth inhibition in a PTEN-independent manner. Sensitive cell lines also exhibited increased expression of EGFR ligands or HER3. In contrast, lapatinib-resistant cell lines exhibited high androgen receptor (AR) levels or epithelial-to-mesenchymal transition (post-EMT) features. In endometrial cancer cells, at a wide range of clinically achievable drug concentrations, additive and synergistic interactions were observed for lapatinib plus carboplatin, paclitaxel, docetaxel, and doxorubicin. These observations provide a clear biologic rational to test lapatinib as a single agent or in combination with chemotherapy in endometrial cancer with HER2 overexpression. Expression of EGFR, its ligands, HER3, AR, and post-EMT markers warrant further evaluation to help define patients with HER2-nonoverexpressing endometrial cancer most likely to benefit from lapatinib.

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Figures

Figure 1
Figure 1
(A) Growth inhibitory effects of lapatinib were studied across a panel of endometrial cancer cell lines. Cells were grown in cell line-specific media without or with increasing doses of lapatinib (ranging between 0.031 and 10 μM). Cells were trypsinised and counted after 7 days of treatment. The percentage of inhibition was calculated compared with untreated controls. The log of the fractional growth inhibition was plotted against the log of the drug concentration. The dose achieving 50% growth inhibition (IC50) was interpolated from the resulting linear regression curve fit. Cell lines are ordered from left to right from low to high IC50 values. Error bars indicate the SE of the mean value. Mean is derived from three replicate experiments. (B) HER2 and topo-II-α gene copy number were analysed by FISH using a multicolor topo-II-alpha spectrum orange, HER2 spectrum green, and CEP17 spectrum aqua probe (Vysis Inc.). (C) Epidermal growth factor receptor and HER2 expression were assessed by ELISA for each of the endometrial cancer cell lines. Epidermal growth factor receptor was correlated with the corresponding IC50 value (Spearman's ρ correlation coefficient). For HER2, the IC50 values were compared between cell lines with HER2 overexpression and those without using the Mann–Whitney test for statistical comparison.
Figure 2
Figure 2
(A) Dose-dependent activity of lapatinib on HER2, EGFR, AKT, and ERK phosphorylation in USPC1, USPC2, RL-95-2, HEC155, and SNG-II cells. Both cell lines were treated with increasing doses of lapatinib (0.5–5 μM) for 1 h. Immunoprecipitation and western blotting to detect phosphorylated HER2 or EGFR were performed as described in Materials and Methods. ERK and AKT phosphorylation levels were detected using phospho-specific ERK and AKT antibodies as described in Materials and Methods. (B) Western blotting to detect PTEN expression was performed as described in Materials and Methods.
Figure 3
Figure 3
(A) The red and green matrices represent the normalised expression patterns for each gene in Table 2 across the 19 endometrial cancer cell lines. Brightest red indicates highest relative expression; brightest green indicates lowest relative expression. The reference cRNA pool consists of equal amounts of cRNA from the 18 endometrial cancer cell lines. Cell lines are ordered from low IC50 values to high IC50 values. Genes are weighted according to their correlation with the IC50 values. A negative correlation indicates that the marker is associated with sensitivity to lapatinib. (B) A cluster diagram of the 19 endometrial cancer cell lines was developed using the markers VIM, CDH1, CDH3, and the KRT5, 6, 8, 18, and 19. Post-EMT cell lines showed high expression of VIM, but low expression of CDH1, CDH3, and KRT8, 18, and 19 (eg, MFE296, HEC1B, AN3CA, EN1, EN, and EFE184).
Figure 4
Figure 4
Mean CI values for chemotherapeutic drug–trastuzumab combinations in four different human endometrial cancer cell lines. Error bars indicate the 95% confidence interval of the mean value. Mean is derived from three replicates spanning clinically relevant concentration ranges sufficient to inhibit growth of control cells by 20–90%. Combination index values were derived from parameters of the median effect plots, and statistical tests were used to determine whether the CI values at multiple effect levels (IC20–IC90) were statistically significantly different from CI values equal to 1. Values that are statistically significantly less than 1 indicate synergistic interactions. Values that are statistically significantly greater than 1 indicate antagonistic interactions. Values equal to (or not statistically significantly different from) 1 indicate additive interactions.
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