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. 2016 Dec 13;7(50):82511-82527.
doi: 10.18632/oncotarget.12733.

IGF-1 Contributes to the Expansion of Melanoma-Initiating Cells Through an Epithelial-Mesenchymal Transition Process

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Free PMC article

IGF-1 Contributes to the Expansion of Melanoma-Initiating Cells Through an Epithelial-Mesenchymal Transition Process

Vincent Le Coz et al. Oncotarget. .
Free PMC article

Abstract

Melanoma is a particularly virulent human cancer, due to its resistance to conventional treatments and high frequency of metastasis. Melanomas contain a fraction of cells, the melanoma-initiating cells (MICs), responsible for tumor propagation and relapse. Identification of the molecular pathways supporting MICs is, therefore, vital for the development of targeted treatments. One factor produced by melanoma cells and their microenvironment, insulin-like growth factor-1 (IGF- 1), is linked to epithelial-mesenchymal transition (EMT) and stemness features in several cancers.We evaluated the effect of IGF-1 on the phenotype and chemoresistance of B16-F10 cells. IGF-1 inhibition in these cells prevented malignant cell proliferation, migration and invasion, and lung colony formation in immunodeficient mice. IGF-1 downregulation also markedly inhibited EMT, with low levels of ZEB1 and mesenchymal markers (N-cadherin, CD44, CD29, CD105) associated with high levels of E-cadherin and MITF, the major regulator of melanocyte differentiation. IGF-1 inhibition greatly reduced stemness features, including the expression of key stem markers (SOX2, Oct-3/4, CD24 and CD133), and the functional characteristics of MICs (melanosphere formation, aldehyde dehydrogenase activity, side population). These features were associated with a high degree of sensitivity to mitoxantrone treatment.In this study, we deciphered new connections between IGF-1 and stemness features and identified IGF-1 as instrumental for maintaining the MIC phenotype. The IGF1/IGF1-R nexus could be targeted for the development of more efficient anti-melanoma treatments. Blocking the IGF-1 pathway would improve the immune response, decrease the metastatic potential of tumor cells and sensitize melanoma cells to conventional treatments.

Keywords: EMT; IGF-1; chemoresistance; melanoma-initiating cells; metastasis.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors have no competing financial interests to declare.

Figures

Figure 1
Figure 1. IGF-1 downregulation impairs lung colony formation by B16-F10 melanoma cells in immunocompetent and immunodeficient mice
(A) IGF-1 levels were analyzed in four IGF-1dull clones (A6, C10, E11, F9), the control B16-F10CT and the parental B16-F10WT cells, by confocal microscopy. Red, IGF-1; Blue, nuclei stained with DAPI. The scale bar represents 10 μm. (B) IGF-1 levels in the various cell lines were analyzed by immunoblotting. β-actin was used as a loading control. Graphs show quantification of the immunoblot signals (IGF-1/β-actin). Data are represented as the mean ± standard error of mean (SEM) for three independent determinations. n.s., not significant, p < 0.05 p < 0.01, p < 0.001 versus B16-F10CT cells. (C–D) Lung colony formation. Groups of C57BL/6 and NSG mice received 1 × 105 cells via injection into the retro-orbital sinus (day 0). Fifteen days after inoculation with cells, the lungs were excised and nodule development was analyzed. Representative images of the lung nodules are shown. Horizontal bars represent the mean number of lung colonies ± SEM per C57BL/6 mouse. Clones E11 (14.5 ± 5.3 nodules), F9 (10.4 ± 5.6 nodules) and C10 (1.6 ± 1.6 nodules) had a significantly lower level of lung colony formation (p < 0.001, n = 5) than clone A6 (62 ± 30.6 nodules), B16-F10CT (73.8 ± 19.2 nodules) and B16-F10WT (73 ± 3.6 nodules) cells. n.s., not significant. In NSG mice, nodule development was quantified as the percentage of the lung occupied by tumors. Images of representative tumors are displayed for each in vivo experiment. The C10 clone formed lung colonies significantly less efficiently than B16-F10CT and B16-F10WT cells (1 ± 0.6% versus 58.3 ± 23 % and 44.1 ± 18.6% of the lung occupied by tumors, respectively, p < 0.05, n = 3).
Figure 2
Figure 2. IGF-1 downregulation impairs cell proliferation
(A) B16-F10 cell proliferation. B16-F10WT, B16-F10CT and C10 cells were plated in 96-well plates and cell proliferation was assessed for 96 h in the MTT assay. The data presented are the mean (±SEM) of quadruplicate experiments. C10 had a lower proliferation rate than control cells, from 48 h onwards (0.08 ± 00.7 versus 0.002 ± 0.012 OD values, p < 0.001, n = 4). (B) Cell cycle analysis. After staining with propidium iodide, the distribution of the cells between the G0/G1, S and G2/M phases was determined by flow cytometry. Top, Representative cell cycle plots from one experiment are shown. The distribution and percentage of cells in the G0/G1, S and G2/M phases of the cell cycle are indicated at the top right of each histogram. Bottom, Data are represented as means ± SEM. IGF-1 downregulation resulted in a lower percentage of cells in S-phase (18.9±1.4%) for C10 cells than for B16-F10WT (36.5 ± 3.7%) and B16-F10CT cells (33.0 ± 2.5%, p < 0.001, n = 4), and a higher percentage of cells in G0/G1 phase in C10 cells (60.4% +/–2.3) than in B16-F10WT (43.9 ± 1.0%) and B16-F10CT cells (46.4 ± 1.7%, p < 0.001, n = 4). (C) Quantification of cyclin D1 and p27 protein levels in B16-F10WT, B16-F10CT and C10 cells. Graphs represent the quantification of immunoblot signals, corresponding to the cyclin D1/β-actin and p27/β-actin ratio. Data are represented as means ± SEM for three independent determinations. (D) Clonogenic assay. B16-F10WT, B16-F10CT and C10 cells were assessed for clonogenic potential. The data presented are the means (±SEM) of quintuplet experiments. C10 colonies were significantly smaller than the colonies for control and parental cells (14 ± 2 colonies with a diameter > 75 μm for the C10 clone versus 241 ± 6 and 223 ± 10 for B16-F10WT and B16-F10CT, respectively, p < 0.02, n = 5).
Figure 3
Figure 3. IGF-1 downregulation promotes mesenchymal-to-epithelial transition
(A) Western blot analysis of an epithelial marker (E-cadherin), a mesenchymal marker (N-cadherin), and an EMT regulator (ZEB1) in C10, B16-F10CT and B16-F10WT cells. β-actin was used as the loading control. Graphs represent the quantification of the immunoblot signals corresponding to the protein of interest/β-actin ratio. Data are represented as means ± SEM for three independent determinations, p < 0.05, p < 0.001 versus B16-F10CT cells. (B) Flow cytometry analysis of surface expression of the mesenchymal markers CD44, CD29 and CD105. The dark-colored bars correspond to cells incubated with the antigen-specific antibody, and gray bars correspond to cells incubated with the isotype-matched control antibody. Mean fluorescence intensity values for each marker are shown at the top left of each histogram. Each panel corresponds to three independent experiments with similar results, p < 0.05, p < 0.001 versus B16-F10CT cells.
Figure 4
Figure 4. IGF-1 downregulation decreases cell invasion and migration
(A) Scratchwound healing assay. Cells were grown to confluence and then used for wound healing migration assays. Artificial wounds were made in confluent monolayers of C10, B16-F10CT or B16-F10WT cells. Top, Migration of melanoma cells towards the wound, photographed after 6 h, 12 h and 24 h. The top panel shows one of three independent experiments. Bottom, Data are shown as means ± SEM. No significant differences in migratory activity were observed between B16-F10WT and B16-F10CT cells, with 67.7 ± 5.4% and 73.3 ± 2.4%, respectively, of the area displaying recovery at 12 h and 83.9 ± 6.3% and 91.6 ± 3.1%, respectively, of the area displaying recovery at 24 h. However, C10 clones displayed lower levels of migratory activity, with only 26.7 ± 5.5% (p < 0.01, n = 3) and 35.9 ± 1.4% (p < 0.001, n = 3) of recovery at 12 h and 24 h, respectively. (B) F-actin expression was analyzed by fluorescence microscopy with phalloidin (green) as a marker of membrane protrusions. The scale bar represents 10 μm. White arrowheads indicate the pseudopods. Each experiment was performed three times. (C) Invasion assay. C10, B16-F10CT and B16-F10WT cells were cultured in Matrigel Transwells. Their invasive potential was analyzed 96 h later, by fluorescence microscopy. The nuclei are stained blue with DAPI. Top, Representative images of three independent experiments are shown. The scale bar represents 50 μm. Bottom, Data are shown as means ± SEM. C10 clones have a lower invasion capacity (54.2 ± 9.2 cells per field) than parental (156.6 ± 8.3 cells per field) and control cells (131.6 ± 12.6 cells per field, p < 0.001, n = 3).
Figure 5
Figure 5. IGF-1 downregulation decreases the expression of cancer stem-like and pluripotency markers in B16-F10 melanoma cells
(A) Flow cytometry analysis of the canonical self-renewal transcription factors Oct-3/4 and SOX2 in the C10 clone, B16-F10CT and B16-F10WT cells. Top, Dark-colored bars correspond to cells incubated with the antigen-specific antibody, and gray bars correspond to cells incubated with the isotype-matched control antibody. Mean fluorescence intensity values for each marker are shown at the top left of each histogram. All panels represent at least four independent experiments showing similar results. Bottom, Data are represented as means ± SEM for three independent determinations, p < 0.05, p < 0.01 versus B16-F10CT cells. (B) Western-blot analysis for MITF in C10, B16-F10CT and B16-F10WT cell lysates. β-actin was used as the loading control. Bottom, Graphs show the quantification of three immunoblot signals corresponding to the protein of interest/β-actin ratio, p < 0.01 versus B16-F10CT cells. (C) Analysis of MITF expression (green) by fluorescence microscopy. Blue, nuclei stained with DAPI. The scale bar represents 10 μm. The panel depicts one of three independent experiments. (D) CD24 and CD133 cell surface markers were analyzed by flow cytometry. Top, Panels represent at least four independent experiments with similar results. The percentages of CD24- and CD133-positive cells are shown at the top right of each histogram. Bottom, Data are represented as means ± SEM for three independent determinations, p < 0.05, p < 0.001 versus B16-F10CT cells.
Figure 6
Figure 6. IGF-1 knockdown affects stemness properties and MIC behavior
(A) Melanosphere formation assay. We assessed the ability of parental B16-F10 cells to produce tumor spheres, by suspending 1x103 cells in a serum-free medium and incubating them at 37°C for 7 days. The total number of spheres produced was compared between groups. The scale bar represents 50 μm. Top, Independent experiments were performed three times and a representative experiment is shown. Bottom, Data are shown as means ± SEM. The melanospheres for the IGF-1-knockdown clone (C10) were significantly smaller and fewer (38.3 ± 6.7 spheres per 1000 cells) than those formed by B16-F10WT (210.6 ± 12.9 spheres per 1000 cells) and B16-F10CT(178.3 ± 14.6 spheres per 1000 cells, p < 0.001, n = 3) cells. (B) ALDH activity. ALDH1A1 activity was analyzed in B16-F10WT, B16-F10CT and C10 cells, by flow cytometry with the Aldefluor reagent kit. ALDH1A1 activity was normalized by taking measurements in the presence and absence of the ALDH1-specific inhibitor, diethylamino-benzaldehyde (DEAB: 10 μM). The top panel depicts one of four experiments. The percentage ALDH-positive cells is shown at the bottom right of each histogram. Bottom, Data are shown as means ± SEM. ALDH activity was much weaker in C10 clones (6.5 ± 9.2%) than in B16-F10WT and B16-F10CT cells, which had high levels of ALDH activity (48.2 ± 19.2% and 46.4 ± 23.9%, p < 0.05, n=4 respectively). (C) ALDH1A1 protein levels, as determined by immunoblotting. β-actin was used as the loading control. Graphs represent the quantification of immunoblot signals, corresponding to the ALDH1A1/β-actin ratio. The data shown are the means ± SEM of three independent determinations, p < 0.05. (D) Side population (SP) analysis. C10, B16-F10CT and B16-F10WT cells were stained with Hoechst 33342 dye in the presence and absence of Ko143 (1 μM), and the SP was analyzed by flow cytometry. Top, Data from at least five independent experiments with similar results. Percentages of SP cells are shown at the top left of each histogram. Bottom, Data are shown as means ± SEM. SP cells represent 1.4 ± 0.4% of all cells for B16-F10WT and B16-F10CT cells, but only 0.3 ± 0.2% of total C10 clone cells (p < 0.01, n = 5). (E) Analysis of ABCG2 expression in cell lysates of C10, B16-F10CT and B16-F10WT. β-actin was used as the loading control. Top, Figures are representative of three independent experiments yielding similar results. Bottom, Data are shown as means ± SEM, p < 0.01 versus B16-F10CT cells.
Figure 7
Figure 7. IGF-1 downregulation sensitizes B16-F10 cells to mitoxantrone
(A) Analysis of the intracellular accumulation of MIT in B16-F10WT, B16-F10CT and C10 cells. Mitoxantrone efflux and ABCG2 activity were determined by flow cytometry measurements of mitoxantrone accumulation in the presence and absence of the ABCG2 inhibitor Ko143 (1 μM). Top, Seven independent experiments were performed, and the results of a single representative experiment are shown here. Percentages of cells displaying MIT efflux are shown at the top left of each histogram. Bottom, Data are shown as means ± SEM. C10 cells were significantly less able to exclude MIT (36.9 ± 9.0%) than B16-F10WT and B16-F10CT cells (77.5 ± 21.2% and 79.9 ± 15.1%, respectively, p < 0.05, n = 7). (B) Cell viability. Cells were treated with different concentrations of mitoxantrone (0.1 to 10 μM) and cell viability was determined at 48 h, in the FDA assay. Top The panel depicts one of four experiments. Percentages of FDA-negative (dead) cells are shown at the top left of each histogram. Bottom, Data are shown as means ± SEM. B16-F10WT and B16-F10CT cells were consistently found to be more resistant to MIT than the C10 clone, from the dose of 2 μM MIT upwards, with 70.8 ± 3.0% cell death for the C10 clone versus 38.3 ± 4.7% and 43.8 ± 4.25% cell death for B16-F10WT and B16-F10CT cells, respectively, p < 0.01, n = 4.

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