Lapatinib Suppresses HER2-Overexpressed Cholangiocarcinoma and Overcomes ABCB1- Mediated Gemcitabine Chemoresistance

Abstract

Background: Recent breakthroughs in cholangiocarcinoma (CCA) genomics have led to the discovery of many unique identifying mutations, of which HER2 has been found to be overexpressed specifically in cases of extrahepatic CCA. However, whether or not lapatinib (an oral tyrosine kinase inhibitor selective for inhibition of HER2), or a combination of lapatinib and gemcitabine, exerts inhibitory effects on HER2-overexpressed CCA is still unclear.

Methods: The effect of lapatinib and a lapatinib-gemcitabine combination treatment on CCA was determined using organoid and cell line models. Cell cycle arrest, apoptosis and proteins involving HER2-dependent downstream signaling pathways were analyzed to assess the effect of lapatinib on HER2⁺ CCA. The synergistic effect of lapatinib and gemcitabine was interpreted by docking analysis, ABCB1-associated ATPase assay, rhodamine transport assay and LC-MS/MS analyses.

Results: dFdCTP, the active metabolite of gemcitabine, is proved to be the substrate of ABCB1 by docking analysis and ATPase assay. The upregulation of ABCB1 after gemcitabine treatment accounts for the resistance of gemcitabine. Lapatinib exerts a dual effect on HER2-overexpressed CCA, suppressing the growth of CCA cells by inhibiting HER2 and HER2-dependent downstream signaling pathways while inhibiting ABCB1 transporter function, allowing for the accumulation of active gemcitabine metabolites within cells.

Conclusions: Our data demonstrates that lapatinib can not only inhibit growth of CCA overexpressing HER2, but can also circumvent ABCB1-mediated chemoresistance after gemcitabine treatment. As such, this provides a preclinical rationale basis for further clinical investigation into the effectiveness of a combination treatment of lapatinib with gemcitabine in HER2-overexpressed CCA.

Keywords: ABCB1; HER2; chemoresistance; cholangiocarcinoma; gemcitabine; lapatinib.

Figure 1

Patient-derived CCA organoids recapitulate the histopathological features of the primary tumors. (A) A schematic representation of CCA organoids collection, processing and experimental designs. (B) H&E staining images of the CCA tissue specimens CC6062, CC2196, CC9630 and the derived organoids. Scale bar, 50 μm. (C) Multiplex immunofluorescence co-staining images of HER2 (red), MUC-1 (green), CK7 (grey) of primary clinical tissue (top row of each group) and organoids (bottom row of each group). Scale bar, 50 μm.

Figure 2

Lapatinib exhibits a stronger inhibitory effect on the growth of HER2-overexpressed CCA patient-derived organoids. (A) Bright-field images of HER2-overexpressed CCA organoids treated with 5 μM lapatinib. Relative numbers (n = 3 biologically independent samples per group) and sizes (n =20 biologically independent organoids per group) of organoids were quantified as fold-change compared to control. Scale bar = 200 μm. (B) Bright-field images of CC6062, CC2196 and CC9630 treated with 5μM lapatinib (Lapa) compared to control. Scale bar, 20μm. (C) Growth inhibitory effect curves of lapatinib (Lapa) in patient-derived CCA organoids proved that CC6062 is more sensitive to lapatinib when compared to other CCA organoids. The data is expressed as the mean ± S.D., two-sided Student’s t-test, ***p < 0.001.

Figure 3

The underlying mechanisms by which lapatinib (Lapa) induces FRH-0201 cell cycle arrest and apoptosis in a concentration- and time-dependent manner. (A, B) HER2 protein expression in CCA cell lines was detected by Western blotting and immunofluorescence. (C, D) Growth inhibitory effects of lapatinib (Lapa) in HER2⁺ and HER2^- cell lines. (E, F) Lapatinib dramatically induced G1 cell cycle arrest in HER2-positive CCA cells in both time and dose-dependent manners. (G, H) Apoptosis of FRH-0201 was induced post-exposure to varying treatment concentrations of lapatinib or a constant concentration exposure over different time durations. (I, J) Down-regulation of c-myc was observed with induction of p27^Kip1 and down-regulation of Cyclin D1 after lapatinib treatment (K, L) HER2 and HER2-dependent downstream signaling pathways were suppressed by lapatinib in FRH-0201 cells. (M) A Schematic representation of the mechanisms with which lapatinib inhibits the growth of HER2⁺ CCA cells. The data is expressed as the mean ± S.D., two-sided Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

Lapatinib exerts synergistic growth inhibitory effects on HER2-overpressed CCA organoid and cell line when combined with gemcitabine. (A) Synergistic growth inhibitory effects of a lapatinib (Lapa) - gemcitabine (Gem) combination treatment of HER2-overexpressed organoids. (B) Bright-field images of HER2-overexpressed tumor organoids treated with DMSO, gemcitabine (5 μM Gem), lapatinib (5 μM Lapa) and gemcitabine+lapatinib(5 μM Gem +5 μM Lapa) for 4 days. Scale bar, 20 μm. (C) Synergistic growth inhibitory effects of lapatinib (Lapa) combined with gemcitabine (Gem) in FRH-0201. (D, E) The lapatinib-gemcitabine combined treatment induced G1 arrest and apoptosis in FRH-0201. Data is expressed as the mean ± S.D. based on three independent experiments.

Figure 5

Lapatinib suppresses the function of elevated ABCB1 after being treated with gemcitabine in CCA cells. (A) The growth inhibitory effects of gemcitabine (Gem) on FRH-0201 and FRH0201-Gem. (B, C) ABCB1 protein expression in FRH-0201 and FRH0201-Gem was detected with Western blotting and immunofluorescence. Scale bar: 20 μm. (D) Quantification of fluorescence intensity for ABCB1 in FRH-0201, FRH0201-Gem and FRH0201-Gem incubated with 10μM lapatinib (Lapa) (E, F) Relative mRNA levels and protein levels of ABCB1 in various CCA cell lines after treatment with gemcitabine (10 μM Gem) or a combination of gemcitabine and lapatinib (10μM Gem +5μM Lapa) for 48 h. (G) Upper left: The panoramic structure of ABCB1 and dFdCTP binding site. Upper right: A detailed three-dimensional plot of the interaction of dFdCTP and ABCB1. Bottom left: The panoramic structure of ABCB1 and lapatinib binding site. Bottom right: A detailed three-dimension plot of the interaction between lapatinib and ABCB1. The ABCB1 protein is depicted in red. H-bondings are shown in purple, hydrophobic bonds are shown in green, and mild polar bonds are shown in blue. Lapatinib and dFdCTP are depicted with the following color codes: carbon (blue), oxygen (red), nitrogen (dark blue), sulfur (yellow), fluoride (green), hydrogen (grey), chlorine (dark green), phosphorus (purple). (H) The luminescence increases in a dose-dependent manner following the incubation of verapamil (positive control), lapatinib or dFdCTP with P-glycoprotein-containing membranes. (I) ABCB1 ATPase activity increases in a dose-dependent manner with varying concentrations of verapamil, lapatinib, and dFdCTP. (J) The fluorescence intensity changes in rhodamine-dyed FRH0201-Gem after treatment with previously indicated concentrations of lapatinib. (K) Confocal images of CC6062 dyed with rhodamine after being treated with or without lapatinib. The data is expressed as the mean ± S.D., two-sided Student’s t-test, n.s. not significant, *p < 0.05, ***p < 0.001.

Figure 6

Lapatinib promotes the accumulation of dFdCTP within CCA cells. (A) Extracted ion chromatography (XIC) and MS/MS spectrum of dFdCTP. (B) Linearity and range of the calibration curve (C) Accuracy and precision of the qualitative method under low, medium and high concentrations. (D) The concentration of intracellular dFdCTP is positively correlated with increasing concentrations of lapatinib. FRH-0201, FRH0201-Gem were treated with a combination of a varying concentrations of lapatinib (0, 1, 5, 20 μM) and a constant concentration of gemcitabine (1 μM). Cells treated with 1 μM Gem + 5 μM Verapamil were set as the positive control. (E) A Schematic representation of the mechanisms with which lapatinib inhibits the efflux of dFdCTP within CCA cells. The data is expressed as the mean ± S.D., two-sided Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001.