Changes in Melanoma Cell Morphology Following Inhibition of Cell Invasion by Third-Generation mTOR Kinase Inhibitors

Abstract

Melanoma is one of the most invasive skin cancers with the highest mortality risk. The PI3K/AKT/mTOR signaling pathways are a key regulatory point related to growth factors and involved in the cell's energy metabolism. They are responsible for cell life processes such as growth, proliferation, invasion, survival, apoptosis, autophagy, and angiogenesis. The studies undertaken concerned the effect of protein kinase inhibitors involved in the signaling pathways of AKT, MEK, and mTOR kinases on the expression of cytoskeletal and extracellular matrix proteins, invasion process, and activities of the matrix metalloproteinases (MMPs): MMP-2 and MMP-9 in melanoma cells. The study used mTOR kinase inhibitors: Everolimus and Torkinib; dual PI3K/mTOR inhibitors BEZ-235 and Omipalisib; and the mTORC1/2 inhibitor OSI-027. These compounds were used both as monotherapy and in combination with the MEK1/2 inhibitor AS-703026. mTOR kinase inhibitors, especially the third generation in combination with the MEK 1/2 kinase inhibitor AS-703026, significantly inhibited invasion and metalloproteinases (MMPs) activity in melanoma cell lines. The inhibition of the cell invasion process was accompanied by a significant change in the expression of proteins associated with EMT. The morphology of cells also changed significantly: their thickness, volume, roughness, convexity of shape, and irregularity, which may be a good diagnostic and prognostic factor for the response to treatment. Our studies to date on the effect of three generations of mTOR kinase inhibitors on the inhibition of the invasion process, the activation of apoptosis, and the reduction in cell proliferation suggest that they may be an important target for anticancer therapy.

Keywords: cell invasion; cell morphology; mTOR protein kinase inhibitors; melanoma.

Figure 1

The effect of mTOR inhibitors on the module In-depth Analysis: “Cell Morphology”: optical thickness avg (µm), optical volume (µm³), roughness avg, shape convexity, irregularity per area (µm²) grown in MEWO melanoma cell line. The experiment was conducted for 72 h. For each inhibitor concentration tested, 178 consecutive time points were measured in 6 independent biological replicates.

Figure 2

(A). The effect of mTOR inhibitors on the module In-depth Analysis: “Cell Morphology”: optical thickness avg (µm) in MEWO melanoma cell lines. The experiment was conducted for 72 h. For each inhibitor concentration tested, 178 consecutive time points were measured in 6 independent biological replicates (B). The effect of mTOR inhibitors on the module In-depth Analysis: “Cell Morphology”: roughness avg and shape convexity in MEWO melanoma cell lines. The experiment was conducted for 72 h. For each inhibitor concentration tested, 178 consecutive time points were measured in 6 independent biological replicates (C). The effect of mTOR inhibitors on the module In-depth Analysis: “Cell Morphology”: irregularity and area (µm²) in MEWO melanoma cell lines. The experiment was conducted for 72 h. For each inhibitor concentration tested, 178 consecutive time points were measured in 6 independent biological replicates.

Figure 2

(A). The effect of mTOR inhibitors on the module In-depth Analysis: “Cell Morphology”: optical thickness avg (µm) in MEWO melanoma cell lines. The experiment was conducted for 72 h. For each inhibitor concentration tested, 178 consecutive time points were measured in 6 independent biological replicates (B). The effect of mTOR inhibitors on the module In-depth Analysis: “Cell Morphology”: roughness avg and shape convexity in MEWO melanoma cell lines. The experiment was conducted for 72 h. For each inhibitor concentration tested, 178 consecutive time points were measured in 6 independent biological replicates (C). The effect of mTOR inhibitors on the module In-depth Analysis: “Cell Morphology”: irregularity and area (µm²) in MEWO melanoma cell lines. The experiment was conducted for 72 h. For each inhibitor concentration tested, 178 consecutive time points were measured in 6 independent biological replicates.

Figure 2

(A). The effect of mTOR inhibitors on the module In-depth Analysis: “Cell Morphology”: optical thickness avg (µm) in MEWO melanoma cell lines. The experiment was conducted for 72 h. For each inhibitor concentration tested, 178 consecutive time points were measured in 6 independent biological replicates (B). The effect of mTOR inhibitors on the module In-depth Analysis: “Cell Morphology”: roughness avg and shape convexity in MEWO melanoma cell lines. The experiment was conducted for 72 h. For each inhibitor concentration tested, 178 consecutive time points were measured in 6 independent biological replicates (C). The effect of mTOR inhibitors on the module In-depth Analysis: “Cell Morphology”: irregularity and area (µm²) in MEWO melanoma cell lines. The experiment was conducted for 72 h. For each inhibitor concentration tested, 178 consecutive time points were measured in 6 independent biological replicates.

Figure 3

The morphological changes in MEWO melanoma cell lines after treatment with mTOR inhibitors for 24 h followed by (A) acridine orange/propidium iodide (AO/PI) microscope objective lens magnification 10×, (B) acridine orange/propidium iodide (AO/PI) magnification 40×, and (C) DAPI staining in the transmitted light magnification 60×. Details regarding the concentrations of the inhibitors are provided in the Materials and Methods section. The experiments were performed in triplicate.

Figure 4

The effect of mTOR inhibitors on in vitro cell invasion melanoma cell lines: MEWO (A), Mel-1359 (B), and WM3211 (C). Cell invasive capacity was assessed via a Matrigel-coated Boyden chamber assay. The accompanying histogram presents the quantification of invasive cells. Data are expressed as the mean ± standard deviation from four technical replicates across two independent experiments. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s post hoc test (Statistica version 12, StatSoft). Levels of statistical significance are denoted as follows: (*) p < 0.05; (**) p < 0.01; (***) p < 0.001. The primary melanoma cell line WM3211 (VGP) has the lowest sensitivity to the applied inhibitors, at 7–22% (Figure 4C).

Figure 5

The effect of mTOR inhibitors on the In-depth Analysis: Wound Healing Assay: (A) gap width; (B) coverage area; (C) representative wound healing assay in MEWO melanoma cell line. The experiment was conducted for 60 (A,B) or 72 h (C), and 178 consecutive time points were measured in 6 independent biological replicates.

Figure 6

The effect of mTOR inhibitors on kinetic motility assays: accumulated mean cell distance; mean cell speed in MEWO melanoma cell line. The experiment was conducted for 60 h. For each inhibitor concentration tested, 178 consecutive time points were measured in 6 independent biological replicates.

Figure 7

The effect of mTOR inhibitors (A) and the combination of mTOR inhibitors with the MEK1/2 inhibitor AS-703026 (B) on gelatinolytic activities of MMP-2 and MMP-9 in MEWO melanoma cell line. Statistical analyses of densitometric activity of MMP-2 and MMP-9 for at least three independent experiments with similar results were performed using one-way analysis of variance with Tukey’s post hoc test (Statistica 12.0 StatSoft); statistical significance at the level of (*) p < 0.05, (**) p < 0.01, (***) p < 0.001.

Figure 8

The effect of mTOR inhibitors (A) and the combination of mTOR inhibitors with the MEK1/2 inhibitor AS-703026 (B) on gelatinolytic activities of MMP-2 and MMP-9 in Mel-1359 melanoma cell line. Statistical analyses of densitometric activity of MMP-2 and MMP-9 for at least three independent experiments with similar results were performed using one-way analysis of variance with Tukey’s post hoc test (Statistica 12.0 StatSoft); statistical significance at the level of (*) p < 0.05, (**) p < 0.01, (***) p < 0.001.

Figure 9

The effect of mTOR inhibitors (A) and the combination of mTOR inhibitors with the MEK1/2 inhibitor AS-703026 (B) on gelatinolytic activities of MMP-2 and MMP-9 in WM3211melanoma cell line. Statistical analyses of densitometric activity of MMP-2 and MMP-9 for at least three independent experiments with similar results were performed using one-way analysis of variance with Tukey’s post hoc test (Statistica 12.0 StatSoft); statistical significance at the level of (*) p < 0.05, (**) p < 0.01, (***) p < 0.001.

Figure 10

The effect of mTOR inhibitors (A) and the combination of mTOR inhibitors with the MEK1/2 inhibitor AS-703026 on the expression of N-cadherin and cytoskeletal and extracellular matrix proteins in MEWO melanoma cell lines. Actin was used as an internal loading control. (B) Densitometric quantification of protein expression was normalized to the corresponding β-actin levels. Presented values correspond to mean ± SD obtained from triplicate experiments. Statistical evaluation was conducted using one-way ANOVA followed by Dunnett’s post hoc test (Statistica 12.0, StatSoft). Statistically significant differences relating to the control group are denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001.