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. 2023 Jan 6;15(2):373.
doi: 10.3390/cancers15020373.

Laminin-332 γ2 Monomeric Chain Promotes Adhesion and Migration of Hepatocellular Carcinoma Cells

Rosanna Scialpi  1 , Valentina Arrè  1 , Gianluigi Giannelli  1 , Francesco Dituri  1
Affiliations

Laminin-332 γ2 Monomeric Chain Promotes Adhesion and Migration of Hepatocellular Carcinoma Cells

Rosanna Scialpi et al. Cancers (Basel). .

Abstract

Extracellular matrix (ECM) has a well-recognized impact on the progression of solid tumors, including hepatocellular carcinoma (HCC). Laminin 332 (Ln332) is a ECM molecule of epithelial basal lamina, composed of three polypeptide chains (α3, β3, and γ2), that is usually poorly expressed in the normal liver but is detected at high levels in HCC. This macromolecule was shown to promote the proliferation, epithelial-to-mesenchymal transition (EMT), and drug resistance of HCC cells. The monomeric γ2 chain is up-regulated and localized preferentially at the invasive edge of metastatic intrahepatic HCC nodules, suggesting its potential involvement in the acquisition of invasive properties of HCC cells. HCC cells were tested in in vitro adhesion, scattering, and transwell migration assays in response to fibronectin and the Ln332 and Ln332 γ2 chains, and the activation status of major signaling pathways involved was evaluated. Here, we show that the Ln332 γ2 chain promotes HCC the cell adhesion, migration, and scattering of HCC cells that express the Ln332 receptor α3β1 integrin, proving to be a causal factor of the EMT program achievement. Moreover, we found that efficient HCC cell adhesion and migration on γ2 require the activation of the small cytosolic GTPase Rac1 and ERKs signaling. These data suggest that the γ2 chain, independently from the full-length Ln332, can contribute to the pro-invasive potential of aggressive HCC cell subpopulations.

Keywords: Gamma 2 subunit; HCC (hepatic cellular carcinoma); cell adhesion; cell migration; cell scattering; laminin 332 (Ln-332); therapeutic target.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Laminin-332 γ2 chain promotes adhesion, scattering, and migration of HCC cells. (a) Cell adhesion assay. HLE or HLF cells were seeded on uncoated (BSA), fibronectin (Fn), laminin-332 (Ln332), or γ2 chain (γ2)-coated well surfaces and allowed to attach and spread for 30 minutes. Data are the means ± SD of triplicates; (b) scattering assay (File S1). Colonies made by 8–10 cells of HLE and HLF cells were left untreated or treated with soluble Ln332 (10 µg/mL) and γ2 (3.125 µg/mL) in serum free medium for 24 h; scale bar = 50 µm; a replicate experiment, including stimulation with hepatocyte growth factor (HGF, at the concentration of 100 ng/mL) as a positive control condition, is shown in Figure S1; (c) transwell migration assay. HLE and HLF cells were seeded on the top of the transwell membrane, previously coated with Fn, Ln332, or γ2 on the lower side and allowed to migrate for 16 h. Data are the means ± SD of five randomly chosen microscopic fields. T test (unpaired, two-tailed), *** p < 0.001 (vs. BSA in panel (a)); and scale bar = 100 µm.
Figure 1
Figure 1
Laminin-332 γ2 chain promotes adhesion, scattering, and migration of HCC cells. (a) Cell adhesion assay. HLE or HLF cells were seeded on uncoated (BSA), fibronectin (Fn), laminin-332 (Ln332), or γ2 chain (γ2)-coated well surfaces and allowed to attach and spread for 30 minutes. Data are the means ± SD of triplicates; (b) scattering assay (File S1). Colonies made by 8–10 cells of HLE and HLF cells were left untreated or treated with soluble Ln332 (10 µg/mL) and γ2 (3.125 µg/mL) in serum free medium for 24 h; scale bar = 50 µm; a replicate experiment, including stimulation with hepatocyte growth factor (HGF, at the concentration of 100 ng/mL) as a positive control condition, is shown in Figure S1; (c) transwell migration assay. HLE and HLF cells were seeded on the top of the transwell membrane, previously coated with Fn, Ln332, or γ2 on the lower side and allowed to migrate for 16 h. Data are the means ± SD of five randomly chosen microscopic fields. T test (unpaired, two-tailed), *** p < 0.001 (vs. BSA in panel (a)); and scale bar = 100 µm.
Figure 2
Figure 2
Adhesion of HCC cells onto γ2 chain of Ln332 promotes activation of pro-migratory signaling mediators. (a) Western blot analysis for active Rac1 (Rac1-GTP) protein levels in HLE and HLF upon seeding and 30 min spreading on uncoated, fibronectin (Fn), Ln332, or γ2-coated surfaces. (b) Western blot analysis for phospho-FAK, phospho-Akt, and phospho-ERKs in HLE and HLF cells upon seeding and 30 min spreading on uncoated, Fn, Ln332, or γ2-coated surfaces. These experiments were performed in replicate (see Figure S2 for row blots).
Figure 3
Figure 3
Adhesion of HCC cells to γ2 chain of Ln332 depends in part on the activation of Rac1. HLE and HLF were pre-incubated with vehicle (DMSO, dilution 1:286 vol/vol), ERK1/2 inhibitor (PD98059, 50 μM), AKT inhibitor (LY294002, 25 μM), or active Rac1 inhibitor (CAS 117786517-6, 50 μM), for 2 h at 37 °C; then, the cells were seeded on uncoated, or fibronectin (Fn), or laminin-332 (Ln332), or γ2 -coated well surfaces and allowed to attach and spread for 30 minutes. Data are the means ± SD of triplicates, T test (unpaired, two-tailed): * p < 0.05, ** p < 0.01, and *** p < 0.001, vs. corresponding ECM protein in control (DMSO).
Figure 4
Figure 4
Migration of HCC cells to fibronectin, Ln332, and Ln332 γ2 chains depends on the activation of ERK1/2. HLE and HLF cells were seeded on the top of the transwell membrane, previously coated with Fn, or Ln332, or γ2, on the lower side and allowed to migrate for 16 h in culture medium serum free with vehicle (DMSO, diluted 1:286 vol/vol), MEK inhibitor (PD98059, 50 μM), AKT inhibitor (LY294002, 25 μM), or active Rac1 inhibitor (CAS 117786517-6, 50 μM). Data are the means ± SD of five randomly chosen microscopic fields. T test (unpaired, two-tailed): ** p < 0.01, and *** p < 0.001 vs. control (DMSO) of related ECM protein coating condition. Scale bar = 100 µm.
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