Galectin-1-mediated MET/AXL signaling enhances sorafenib resistance in hepatocellular carcinoma by escaping ferroptosis

Abstract

Sorafenib, a small-molecule inhibitor targeting several tyrosine kinase pathways, is the standard treatment for advanced hepatocellular carcinoma (HCC). However, not all patients with HCC respond well to sorafenib, and 30% of patients develop resistance to sorafenib after short-term treatment. Galectin-1 modulates cell-cell and cell-matrix interactions and plays a crucial role in HCC progression. However, whether Galectin-1 regulates receptor tyrosine kinases by sensitizing HCC to sorafenib remains unclear. Herein, we established a sorafenib-resistant HCC cell line (Huh-7/SR) and determined that Galectin-1 expression was significantly higher in Huh-7/SR cells than in parent cells. Galectin-1 knockdown reduced sorafenib resistance in Huh-7/SR cells, whereas Galectin-1 overexpression in Huh-7 cells increased sorafenib resistance. Galectin-1 regulated ferroptosis by inhibiting excessive lipid peroxidation, protecting sorafenib-resistant HCC cells from sorafenib-mediated ferroptosis. Galectin-1 expression was positively correlated with poor prognostic outcomes for HCC patients. Galectin-1 overexpression promoted the phosphorylation of AXL receptor tyrosine kinase (AXL) and MET proto-oncogene, receptor tyrosine kinase (MET) signaling, which increased sorafenib resistance. MET and AXL were highly expressed in patients with HCC, and AXL expression was positively correlated with Galectin-1 expression. These findings indicate that Galectin-1 regulates sorafenib resistance in HCC cells through AXL and MET signaling. Consequently, Galectin-1 is a promising therapeutic target for reducing sorafenib resistance and sorafenib-mediated ferroptosis in patients with HCC.

Keywords: Galectin-1; ferroptosis; hepatocellular carcinoma; sorafenib resistance.

Figure 1

Galectin-1 expression is associated with patient survival and sorafenib response in HCC cells. (A) Sorafenib-resistant Huh-7 (Huh-7/SR) cells were established and exposed to various sorafenib doses for 48 and 72 h, and cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (B) The mRNA expression of the Galectin family was examined in Huh-7 and Huh-7/SR cells through qRT-PCR. (C) Correlation between Galectin-1 expression and IC₅₀ of sorafenib in a panel of HCC cells. Pearson’s correlation r = 0.5881, and P = 0.0443. (D) Immunohistochemistry staining was performed to examine Galectin-1 expression in normal liver tissue and HCC tissues. (E) The Oncomine database was analyzed to evaluate Galectin-1 expression in HCC tissues compared with normal tissues, with statistics from individual studies obtained from the Oncomine database; fold change (Log2 median-centered intensity) and P values are presented within the box plot. (F) Evaluation of Galectin-1 expression in different HCC stages by using The Cancer Genome Atlas database (http://ualcan.path.uab.edu/index.html). (G) Kaplan–Meier plot revealing the association of Galectin-1 with overall survival in patients with HCC. High Galectin-1 expression was associated with poor survival in patients with HCC. Hazard ratios and P values are presented within the box plot. (H) Analysis of HCC cells of different tumor grades revealed that high Galectin-1 expression was associated with low overall survival. Hazard ratios and P values are presented within the box plot. Data are representative of at least three independent experiments performed in triplicate. Data are presented as means ± standard deviations. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student’s t test).

Figure 2

Enhanced sorafenib resistance and suppressed sorafenib-mediated ferroptosis in HCC cells with Galectin-1 overexpression. (A, B) Analysis of Galectin-1 expression in Huh-7, Huh-7/SR, vector control (Huh-7/Ctrl), and Galectin-1 overexpressing Huh-7 (Huh-7/Gal-1) cells through Western blotting and qRT-PCR. (C) Cell viability of HCC cells treated with various doses of sorafenib was examined using the MTT assay. (D) Analysis of cancer stem cell marker (Oct-4, Nanog, SOX-2, and KLF4) expression through qRT-PCR and Western blotting. (E) Detection of Galectin-1, glutathione peroxidase 4 (GPX4), and FTH-1 expression in Galectin-1-overexpressing cells, cells treated with sorafenib (10 μM) for 48 h, and cells not treated with sorafenib. (F) Lipid peroxidation detected using the malondialdehyde (MDA) assay. Data are presented as means ± standard deviations. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student’s t test).

Figure 3

Loss of Galectin-1 overcomes sorafenib sensitivity and promotes ferroptosis in HCC cells. (A, B) Stable Galectin-1-silenced Huh-7/SR (Huh-7/SR/shGal#23 and #24) and control (Huh-7/SR/shCtrl) cells were analyzed using Western blotting and qRT-PCR. (C) Sorafenib sensitivity in indicated cells analyzed for 48 and 72 h by using the MTT assay. (D) qRT-PCR and Western blotting were used to determine the expression of cancer stem cells markers (Oct-4, Nanog, SOX-2, and KLF4). (E) Western blotting analysis of Galectin-1, GPX4, and FTH-1 expression in Galectin-1-knockdown sorafenib-resistant HCC cells, cells treated with sorafenib (10 μM) for 48 h, and cells not treated with sorafenib, respectively. (F) Lipid peroxidation determined using the MDA assay. Data are presented as means ± standard deviations. *P < 0.05 and **P < 0.01 for Huh-7; #P < 0.05, ##P < 0.01, and ###P < 0.001 for Huh-7/SR/shCtrl (Student’s t test).

Figure 4

MET and AXL signaling involved in Galectin-1-mediated sorafenib resistance and ferroptosis in HCC. (A) Galectin-1-knocked-down Huh-7/SR (Huh-7/SR/shGal#23 and #24) and control (Huh-7/SR/shCtrl) cells were analyzed for RTK expression (EGFR, MET, AXL, and insulin receptor) through Western blotting. Huh-7/SR cells were treated with an MET inhibitor and AXL inhibitor R428 for 48 hr. Analysis of Galectin-1, AXL, and phospho-AXL expression (B upper panel) and MET and phospho-MET expression (C upper panel) were performed using Western blotting. Cell viability of Huh-7/SR cells cotreated with 20 μM of sorafenib and AXL (B lower panel) or an MET inhibitor (C lower panel) for 48 h. Galectin-1 overexpression after treatment with the MET inhibitor and AXL inhibitor R428 for 48 h. Analysis of AXL and phospho-AXL expression (D upper panel) and MET and phospho-MET expression (E upper panel) by using Western blotting. Cell viability of Galectin-1- overexpressing cells cotreated with 20 μM of sorafenib and AXL (D lower panel) or a MET inhibitor (E lower panel) for 48 h. (F) Oct-4, Nanog, SOX-2, and KLF4 mRNA expression determined using qRT-PCR in Galectin-1-overexpressing cells treated with an MET inhibitor and an AXL inhibitor R428. Western blot analysis was used to detect AXL, phospho-AXL, GPX4, and ferritin heavy chain 1 expression in cells cotreated with 20 μM of sorafenib and an (G) AXL inhibitor or (H) MET inhibitor. Data are presented as means ± standard deviations. **P < 0.01, and ***P < 0.001 (Student’s t test).

Figure 5

AXL and MET expression was positively associated with HCC. (A) AXL expression was positively correlated with HCC tissues and cirrhosis in the analysis of the Oncomine database (https://www.oncomine.com/). (B) MET expression was significantly upregulated in HCC tissues compared with normal tissues according to the analysis of data in the Oncomine database. (C) Kaplan–Meier plot presenting the association of AXL with HCC. High expression of AXL was associated with poor overall survival in patients with HCC. (D) Kaplan–Meier plot presenting the association of MET with overall survival in patients with HCC. High expression of MET was associated with poor progression-free survival and relapse-free survival in patients with HCC.

Figure 6

Schematic for Galectin-1-mediated AXL/MET signaling in sorafenib resistance in HCC cells. Galectin-1 was significantly overexpressed in Huh-7/SR cells and promoted MET and AXL phosphorylation, contributing to sorafenib resistance and decreased sorafenib-mediated ferroptosis. Combined treatment with sorafenib and the AXL/MET inhibitor blocked Galectin-1-mediated AXL/MET signaling, overcoming sorafenib resistance and sorafenib-mediated ferroptosis in Huh-7/SR cells.