Cancer Res Treat.  2021 Apr;53(2):457-470. 10.4143/crt.2020.585.

Effect of Combining EGFR Tyrosine Kinase Inhibitors and Cytotoxic Agents on Cholangiocarcinoma Cells

• Boonsri B
• Yacqub-Usman K
• Thintharua P
• Myint KZ
• Sae-Lao T
• Collier P
• Suriyonplengsaeng C
• Larbcharoensub N
• Balasubramanian B
• Venkatraman S
• Egbuniwe IU
• Gomez D
• Mukherjee A
• Kumkate S
• Janvilisri T
• Zaitoun AM
• Kuakpaetoon T
• Tohtong R
• Grabowska AM
• Bates DO
• Wongprasert K

• KMID: 2514927
• DOI: http://doi.org/10.4143/crt.2020.585

Abstract

Purpose
The potential of members of the epidermal growth factor receptor (ErbB) family as drug targets in cholangiocarcinoma (CCA) has not been extensively addressed. Although phase III clinical trials showed no survival benefits of erlotinib in patients with advanced CCA, the outcome of the standard-of-care chemotherapy treatment for CCA, gemcitabine/cisplatin, is discouraging so we determined the effect of other ErbB receptor inhibitors alone or in conjunction with chemotherapy in CCA cells. Materials and Methods ErbB receptor expression was determined in CCA patient tissues by immunohistochemistry and digital-droplet polymerase chain reaction, and in primary cells and cell lines by immunoblot. Effects on cell viability and cell cycle distribution of combination therapy using ErbB inhibitors with chemotherapeutic drugs was carried out in CCA cell lines. 3D culture of primary CCA cells was then adopted to evaluate the drug effect in a setting that more closely resembles in vivo cell environments.
Results
CCA tumors showed higher expression of all ErbB receptors compared with resection margins. Primary and CCA cell lines had variable expression of erbB receptors. CCA cell lines showed decreased cell viability when treated with chemotherapeutic drugs (gemcitabine and 5-fluorouracil) but also with ErbB inhibitors, particularly afatinib, and with a combination. Sequential treatment of gemcitabine with afatinib was particularly effective. Co-culture of CCA primary cells with cancer-associated fibroblasts decreased sensitivity to chemotherapies, but sensitized to afatinib. Conclusion Afatinib is a potential epidermal growth factor receptor targeted drug for CCA treatment and sequential treatment schedule of gemcitabine and afatinib could be explored in CCA patients.

Keyword

Cholangiocarcinoma; ErbB receptors; ErbB targeted drugs; Antineoplastic agents; Gemcitabine; Afatinib

Figure

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  Fig. 1 Expression of ErbB receptors in surgically resected patient cholangiocarcinoma (CCA) tissue. (A) Absolute RNA expression of epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER) 2, HER3, and HER4 transcript levels in paired CCA patient resected tissue and adjacent margin, detected by droplet digital polymerase chain reaction (ddPCR). Significant difference was determined by paired t test. (B) Absolute expression of EGFR, HER2, HER3, and HER4 transcript levels in primary CCA cells isolated from the tumor tissue and primary cells from normal tissue margins, detected by ddPCR. Significant difference was determined by Welch’s t test. *p < 0.05, **p < 0.01, ***p < 0.001 compared with margin. (C) Immunohistochemistry for ErbB proteins in CCA tissues. EGFR: placenta (positive control EGFR); breast cancer (positive control HER2 and HER3) and normal liver (positive control HER4). Immunopositive cells stained with 3,3′-diaminobenzidine (brown) and counterstained with hematoxylin (blue). Asterisks indicate tumor margin. Scale bars=50 μm.

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  Fig. 2 The percentage of cell survival of different four cholangiocarcinoma cell lines treated with various concentrations of ErbB inhibitors at 48 hours by MTT assay. Afatinib (A), lapatinib (B), and erlotinib (C). The percentage cell survival of the combined treatments of fixed concentration of gemcitabine (0.1 μM) with afatinib, lapatinib, and erlotinib (0.1, 1, and 10 μM) in HuCCA-1 cells (D) and KKU-M213 (E). *p < 0.05 compared with untreated control group, #p < 0.05 compared with the gemcitabine-treated group The combination effect is described as synergism (blue), or antagonism (red) according to the combination index value.

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  Fig. 3 The percentage cell survival of HuCCA-1 (A) and KKU-M213 (B) cells treatment with gemcitabine (0.1 μM), afatinib (0.1 μM blue, 1 μM red, 10 μM green), concurrent treatment of gemcitabine (0.1 μM) and afatinib (0.1, 1, and 10 μM), sequential treatment of gemcitabine (0.1 μM) (6 hours) followed with afatinib (0.1, 1, and 10 μM) (18 hours) and sequential treatment of afatinib (0.1, 1, and 10 μM) (6 hours) followed with gemcitabine (0.1 μM) (18 hours) of survival. *p < 0.05 compared with untreated control group, #p < 0.05 compared with the gemcitabine-treated group, †p < 0.05 compared with afatinib treated group at respective dose of drug.

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  Fig. 4 Effect of gemcitabine and afatinib combination on cell cycle distribution in HuCCA-1 (A) and KKU-M213 (B) cells. Representative Western blotting showing the expression of cyclin D1 and cyclin E proteins in HuCCA-1 (C) and KKU-M213 (F) cells treated with gemcitabine (0.1 μM), afatinib (0.1 μM), concurrent treatment of gemcitabine and afatinib (G+A), sequential treatment of gemcitabine (6 hours) followed with afatinib (18 hours) (G→A) and sequential treatment of afatinib (6 hours) followed with gemcitabine (18 hours) (A→G). Corresponded densitometric analysis of cyclin D1 (D, G) and cyclin E (E, H) proteins expression normalized with α-tubulin in HuCCA-1 and KKU-M213 cells, respectively. Expression value is expressed as fold of control (mean±standard error of mean) from three independent experiment. *p < 0.05 compared with untreated control group, #p < 0.05 compared with the gemcitabine-treated group.

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  Fig. 5 Effect of ErbB inhibitors on primary cholangiocarcinoma (CCA) lines in 3D-tumor growth assay (TGA). (A) Four primary cells were derived from surgically resected CCA tissue samples; CCA-UK5, CCA-UK6, CCA-UK7, and CCA-UK9. Primary cells (dashed), matched cancer associated fibroblasts (CAFs) also derived from the same tissue samples (solid), a human mesenchymal cell line (mesenchymal stem cell [MSC] not shown) or a mixture (CAF+cancer cells shown as dotted line) were employed for the 3D-TGA. (B) Growth of cells, CAFs, and primary cells and CAF (red line) or MSC (green line) mixed with the primary cells was measured using AlamarBlue. Percent cell survival of CCA1 and CCA2 treated with gemcitabine/cisplatin (Gem/Cis) (C), erlotinib (D), or afatinib (E). The mean±20% peak serum concentration for patients is given as the dotted and shaded lines (grey).

Reference

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