Synergism of the receptor tyrosine kinase Axl with ErbB receptors mediates resistance to regorafenib in hepatocellular carcinoma

Abstract

Introduction: Hepatocellular carcinoma (HCC) patients at advanced stages receive immunotherapy or treatment with tyrosine kinase inhibitors (TKIs) such as Sorafenib (Sora) or Lenvatinib in frontline as well as Regorafenib (Rego) or Cabozantinib in second-line. A major hindrance of TKI therapies is the development of resistance, which renders drug treatment futile and results in HCC progression.

Methods: In this study, we addressed the impact of the receptor tyrosine kinase Axl binding to its ligand Gas6 in acquiring refractoriness to TKIs. The initial responses of Axl-positive and Axl-negative cell lines to different TKIs were assessed. Upon inducing resistance, RNA-Seq, gain- and loss-of-function studies were applied to understand and intervene with the molecular basis of refractoriness. Secretome analysis was performed to identify potential biomarkers of resistance.

Results: We show that HCC cells exhibiting a mesenchymal-like phenotype were less sensitive to drug treatment, linking TKI resistance to changes in epithelial plasticity. Gas6/Axl expression and activation were upregulated in Rego-resistant HCC cells together with the induction of ErbB receptors, whereas HCC cells lacking Axl failed to stimulate ErbBs. Treatment of Rego-insensitive HCC cells with the pan-ErbB family inhibitor Afatinib rather than with Erlotinib blocking ErbB1 reduced cell viability and clonogenicity. Genetic intervention with ErbB2-4 but not ErbB1 confirmed their crucial involvement in refractoriness to Rego. Furthermore, Rego-resistant HCC cells secreted basic fibroblast growth factor (bFGF) depending on Axl expression. HCC patients treated with Sora in first-line and with Rego in second-line displayed elevated serum levels of bFGF, emphasizing bFGF as a predictive biomarker of TKI treatment.

Discussion: Together, these data suggest that the inhibition of ErbBs is synthetic lethal with Rego in Axl-expressing HCC cells, showing a novel vulnerability of HCC.

Keywords: Axl; ErbB; GAS6; Regorafenib; bFGF; hepatocellular carcinoma; tyrosine kinase inhibitors.

Figure 1

Axl modulates cellular response to TKIs. (A) Heat map of IC₅₀ values showing sensitivities of HCC cells against TKIs. (B) Release of sAxl and Gas6 into supernatants of HCC cells as determined by ELISA. (C) Heat map of IC₅₀ values indicating sensitivities of parental and Axl-deficient HCC cells against TKIs. (D) Model depicting the generation of TKI-resistant cells. (E) Relative response of parental (ctrl, control, naïve) and resistant HCC cells against TKIs in Axl-proficient and Axl-deficient background. IC₅₀ values were calculated and normalized to sensitive control. Sensitivity of the control was set to the value of 1. (F) Release of Gas6 into supernatants of control (naïve) and TKI-resistant cells as determined by ELISA. (G, H) Transcript and protein levels of Axl in control (naïve) and TKI-resistant HCC cells as analyzed by qPCR (G) and Western Blotting (H), respectively. (I) Expression of Axl (green) in control (naïve) and Sora-, Rego- and Cabo-resistant HCC cells as examined by immunofluorescence microscopy. Cell nuclei were stained with DAPI (blue). White arrows show Axl expression on cell membranes. Data are expressed as mean +/- SD. ns: p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. TKI, tyrosine kinase inhibitor; Sora, Sorafenib; Rego, Regorafenib; Cabo, Cabozantinib; sAxl, soluble Axl.

Figure 2

Axl expression associates with induction of ErbB4 levels in Rego-resistant HCC cells. (A) Heat map of differentially expressed transcripts in control (naïve; light blue bar) vs. Rego-resistant SNU449 cells (dark blue bar). Heat map colors range from blue to red for low to high relative expression (see color gradient at bottom). Arrow indicates the row showing ErbB4 expression in Rego-resistant HCC cells. (B) Axl-dependent genes associating with Rego resistance were identified by overlapping differentially expressed genes of control vs Rego-resistant cell comparisons in an Axl-deficient (green) or Axl-proficient (grey) background. (C) Log₂FC of ErbB4 expression in Rego-resistant versus control cells based on RNA-Seq data. (D) Relative mRNA expression of ErbB4 in control and Rego-resistant SNU449 and SNU449-Axl^- as determined by qPCR. Data are expressed as mean +/- SD. (E) GSEA shows enrichment of ErbB signaling in Rego-resistant SNU449 cells. ns: p > 0.05; **p ≤ 0.01. GSEA, Geneset enrichment analysis; Rego, Regorafenib; FC, fold change.

Figure 3

Axl-dependent activation of ErbB receptors in Rego-resistant HCC cells. (A, B) Quantification of phospho-RTK signals comparing control (ctrl, naïve) and Rego-resistant SNU449 cells in the Axl-proficient (A) and Axl-deficient background (B). Signal intensities of phospho-RTKs were normalized to intensities of controls. Grey bars depict targets of Regorafenib. Arrows indicate Axl and members of the ErbB receptor family and black lines indicate unchanged phosphorylation levels (A, B). (C, D) Relative abundance of selected phospho-RTKs in control (naïve) vs. Rego-resistant SNU449 cells in the Axl-positive (C) and Axl-negative background (D). Data are expressed as mean +/- SD. ns: p > 0.05; **p ≤ 0.01; ***p ≤ 0.001. RTK, receptor tyrosine kinase; Rego, Regorafenib.

Figure 4

Resistance to Rego sensitizes Axl-expressing cells towards Afatinib. (A, B) Clonogenic growth behavior of control (naïve) and Rego-resistant SNU449 cells untreated (w/o) or treated with increasing concentrations of Afatinib in the Axl-proficient (A) and Axl-deficient background (B). Bar charts depict the quantification of colonies relative to control (right panels). (C, D) Dose-response curves of control and Rego-resistant SNU449 cells against Afatinib in the Axl-positive (C) and Axl-negative background (D). Bar charts show sensitivity (IC₅₀) against Afatinib relative to controls (C, D). (A–D) Sensitivities of controls were set to the value of 1. Data are expressed as mean +/- SD. ns: p > 0.05; *p ≤ 0.05; **p ≤ 0.01; Rego, Regorafenib.

Figure 5

Intervention with ErbB2-4 expression re-sensitizes Rego-resistant HCC cells. (A-E) Dose-response curves of Rego-resistant SNU449 cells against Regorafenib upon siRNA-mediated knockdown of EGFR (A), ErbB2 (B), ErbB3 (C), ErbB4 (D) and Axl (E). Bar charts show the respective IC₅₀ values (µM). Data are expressed as mean +/- SD. ns: p > 0.05; *p ≤ 0.05. NT, Non-Target.

Figure 6

Resistance to Rego induces bFGF secretion. (A, B) Quantification of growth factor signals comparing control (ctrl, naïve) and Rego-resistant SNU449 cells in the Axl-proficient (A) and Axl-deficient background (B). Bar charts depicts relative abundance of growth factors to their respective naïve control. Black lines indicate unchanged growth factor levels (A, B). (C) Levels of bFGF in sera of naïve and 2^nd line Rego-treated HCC patients and supernatants of control (white) and Rego-resistant SNU449 cells as determined by ELISA. (D) bFGF levels in supernatants of control (white) and Sora- and Rego-resistant SNU449-Axl^- cells (grey, black) as determined by ELISA. Data are expressed as mean +/- SD. ns: p > 0.05; *p < 0.05. ***p ≤ 0.001. Rego, Regorafenib; Sora, Sorafenib.

Figure 7

Schematic representation of the resistance mechanism of HCC cells to Rego. Axl and ErbB2-4 are co-upregulated in HCC cells resistant to Rego. bFGF levels increase upon acquiring resistance to Rego.