Research ArticleCancer

Adipocytes sensitize melanoma cells to environmental TGF-β cues by repressing the expression of miR-211

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Science Signaling  23 Jul 2019:
Vol. 12, Issue 591, eaav6847
DOI: 10.1126/scisignal.aav6847
  • Fig. 1 Subcutaneous adipocytes are observed in proximity to in situ melanoma.

    (A) Hematoxylin and eosin (H&E) staining of representative vertical cross-sections of melanoma patient samples (n = 5) categorized by Clark’s staging system (stages II to V) and a melanoma metastasis in the lymph node. Scale bars, 2 mm (stages II to IV and lymph) and 5 mm (stage V). Black dashed lines indicate epidermal-dermal junctions; blue lines indicate tumor margins. T, tumor; AD, adipocytes. (B) H&E staining of typical vertical cross-sections from a healthy skin sample (left) and from two melanoma patient samples at different progression stages (right). Scale bar, 2 mm. Arrows indicate adipocytes. Graph plots the mean (±SEM) distance of the adipocytes from the epidermal basal layer from three independent experiments. *P < 0.05, t test. (C) Immunofluorescence analysis of consecutive slices from similar sections described and shown in (B) with melanoma marker HMB-45 (red) and adipocyte-specific marker Plin1 (green). DAPI-stained nuclei appear in blue. Scale bar, 2 mm. (D) Additional immunofluorescence analysis of the patient melanoma sections, represented with the in situ section shown in (C), with fibroblast marker FSP1 (pink) as well as HMB-45 (red) and Plin (green). Scale bars, 50 μm (top) and 20 μm (inset/bottom).

  • Fig. 2 Subcutaneous adipocytes in proximity to in situ melanoma correlate with advanced disease.

    (A) Top: H&E staining of representative vertical cross-sections from four in situ melanoma patient samples with hypodermal adipocytes (left) and five with dermal adipocytes (right). Scale bar, 2 mm. Bottom: Immunofluorescence staining for melanoma marker HMB-45 (red) and adipocyte-specific marker Plin1 (green) in sections from the same patients as atop. DAPI-stained nuclei appear in blue. White dashed line demarcates the basal layer of the epidermis; arrows indicate adipocytes. Scale bar, 200 μm. (B) Immunohistochemical staining for Melan-A in tissue sections from the same patients as in (A). Red circles depict irregular melanocytic nests. Scale bar, 2 mm. (C) Graphs plot the mean number (±SEM) of melanocytic nests (left) and mean size of the nests (μm2; right) in melanoma samples that displayed dermal adipocytes compared to melanoma samples that displayed hypodermal adipocytes. n ≥ 4. *P < 0.05, t test.

  • Fig. 3 Adipocytes drive melanoma plasticity in a reversible manner by miR-211 repression.

    (A) Scheme of experimental design coculture and reverse coculture assays. (B to D) Analysis of migration (B), invasion (C), and XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] proliferation (D) assays using WM3682 melanoma cells in the indicated conditions, described in (A). Images are representative. Scale bars, 100 μm (B) and 50 μm (C) (DAPI-stained nuclei are blue). Graphs plot means (B, at the 24-hour time point; D, relative to sample at time 0) ± SEM from three or more independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.005, t test. (E) Representative Ki67 immunostaining (green) and DAPI nuclear staining (blue) in WM3682 cells in the indicated conditions, described in (A). Scale bars, 50 μm. Graph plots mean ± SEM from three independent experiments; statistical analyses as in (B) to (D). (F) Volcano plot representing the distribution of significantly (fold >1.5, P < 0.05 by t test) differentially expressed miRNAs, noting miR-211, between cocultured and monocultured WM3682 melanoma cells in two independent gene profiling experiments. (G) qRT-PCR analysis of mature and pre-miR-211 expression in WM3682 melanoma cells grown under the indicated conditions, described in (A). Graph plots mean ± SEM from three independent experiments; statistical analyses as in (B) to (D). (H) Representative in vivo bioluminescent images of mice injected with WM1716 cells stably expressing miR-211 or a scrambled control at day 70 after injection. Graph plots mean ± SEM at the indicated time points from five mice per group; statistical analyses as in (B) to (D). (I) Representative ex vivo bioluminescence images of local xenografts, livers, and lungs isolated from mice injected with WM1716 cells stably transfected with miR-211 or scrambled control at 70 days after injection. Graph shows mean bioluminescence ± SEM from four mice per group; statistical analyses as in (B) to (D). (J) Schematic of experimental design to assess the effects of miR-211 expression in melanoma cells cultured with or without adipocyte-conditioned medium. (K to M) Migration (K), invasion (L), and proliferation (M) assays using WM3682 melanoma cells in the indicated conditions, described in (J). Scale bars, 100 μm (K) and 50 μm (L). Graphs, n, and statistical analyses are as described in (B) to (D).

  • Fig. 4 Adipocytes decrease miR-211 expression through secretion of IL-6 and TNF-α.

    (A) Amount of IL-6 in medium conditioned by adipocytes compared to that in control medium. Data are means ± SEM from three independent experiments. ***P < 0.005, t test. (B to D) Analysis of miR-211 expression by qRT-PCR (B), invasion (C), and proliferation by XTT assay (D) in WM3682 melanoma cells treated for 48 hours with IL-6 (40 ng/ml) or TNF-α (15 ng/ml) relative to each in vehicle-treated cells. Images (C) are representative; DAPI-stained nuclei are blue. Scale bar, 50 μm. Relative proliferation (D) was assessed at day 3. Data are means ± SEM from three independent experiments. *P < 0.05 and ***P < 0.005, t test. (E to H) Experimental schematic (E) used to assess miR-211 expression by qRT-PCR (F), invasion (G), and relative proliferation by XTT assay (H) in WM3682 melanoma cells cultured in adipocyte-conditioned medium and treated with an IL-6 inhibitor (IL-6-i) (cucurbitacin I, 0.1 μM) or a TNF-α inhibitor (TNF-α-i) (R7050, 0.5 μM) for 48 hours. Graph plots, n, statistical analyses, images, and scale bars are as described in (B) to (D). (I) Representative images and analysis of healthy donor normal skin and in situ melanoma patient sections (examined in proximity of adipocytes or not) immunofluorescently stained for melanoma marker HMB-45 (pink), IL-6 (red), and adipocyte-specific marker Plin1 (green) and counterstained with DAPI (blue). Scale bars, 50 μm (top) and 20 μm (insets/bottom). Arrows indicate IL-6. (J) Kaplan-Meier survival plot of patients bearing melanoma with no overexpression (blue) or with overexpression of the IL-6 receptor (IL-6R) (red). Data were obtained from the Cancer Genome Atlas (n = 550; P = 0.00971).

  • Fig. 5 miR-211 attenuates TGF-β signaling and reduces the sensitivity of melanoma cells to TGF-β.

    (A) Cell signaling pathway using GSEA analysis based on transcriptome profiling of WM1716 melanoma cells stably expressing miR-211 or scrambled control. TGF-β pathway score, P < 0.0056; down-regulated TGF-β genes, P < 0.037; up-regulated TGF-β genes, P < 0.157. See also data files S2 and S3. (B) Luciferase activity assay of a TGF-β–responsive reporter in WM1716 and WM3314 cells stably expressing miR-211 or a scrambled control. Data are means ± SEM from three independent experiments. ***P < 0.005, t test. (C) Luciferase activity assay of TGF-β–responsive reporter in WM3682 cells cotransfected with antagomiR–miR-211 or a scrambled control and either siSMAD4 or scrambled siRNA. Data are means ± SEM from three independent experiments; statistical analyses as in (B). (D and E) Western blot analysis of phosphorylated and total SMAD2 abundance (D) and SMAD4 immunostaining (red; E) in the indicated cells. β-Actin served as loading control (D); DAPI counterstained the nuclei (blue; E). Scale bar, 20 μm. (F) qRT-PCR analysis of the expression of a panel of TGF-β signaling–related genes in WM3314 cells stably expressing miR-211 relative to those expressing a scrambled control. Data are means ± SEM from four independent experiments. *P < 0.05 and ***P < 0.005, t test. (G) Luciferase activity assay of TGF-β–responsive reporter in the indicated cells upon treatment with recombinant TGF-β (2 ng/ml) or dimethyl sulfoxide (DMSO) (ctrl). Data are means ± SEM from three independent experiments; statistical analyses as in (F). (H) XTT proliferation assay of cells described and treated as in (G). Data are mean fold change relative to day 0 ± SEM from three independent experiments; statistical analyses as in (F). (I) Venn diagram showing the overlap between the top 20% of down-regulated genes in WM1716 melanoma cells upon miR-211 introduction, the miR-211–predicted targets, and the genes identified as involved in TGF-β signaling (n = 2). (J) qRT-PCR analysis of TGFBR1 mRNA expression in the indicated cells. Data are means ± SEM from three independent experiments; statistical analyses as in (B). (K) Western blot analysis of TGFBR1 protein abundance in the indicated cells. β-Tubulin served as a loading control. Blots are representative of three independent experiments. (L) Predicted miR-211 target site identified in TGFBR1 3′ UTR (red). Wild-type (WT) and mutated (MUT) miR-211 binding site sequences. (M) Luciferase activity assay of WT or MUT TGFBR1 3′ UTR reporter constructs in WM3314 and WM1716 cells stably expressing miR-211 or scrambled control. Graphs and statistical analyses are as described in (G). (N) Luciferase activity assay of TGF-β–responsive reporter in WM3314 and WM1716 cells stably expressing miR-211 or scrambled control, which were transfected with either TGFBR1 complementary DNA (cDNA) that lacks the 3′ UTR or an empty vector (ctrl). Data are means ± SEM from three independent experiments. **P < 0.01 and ***P < 0.005, t test.

  • Fig. 6 Adipocytes sensitize melanoma cells to TGF-β by repressing miR-211 expression.

    (A) Western blot analysis of phospho-SMAD2 (pSMAD2) and SMAD2 (top) and TGFBR1 (bottom) protein expression in WM3682 melanoma cells in indicated conditions (see Fig. 2A for experimental scheme). β-Actin and β-tubulin served as loading controls. (B) Luciferase activity assay of TGF-β signaling–related genes in WM3682 cells cocultured or reverse cocultured with adipocytes relative to monocultured cells. Data are means ± SEM from three independent experiments; *P < 0.05 and ***P < 0.005. (C) Experimental schematic in (D) to (G). (D and E) Luciferase activity of a TGF-β–responsive reporter in WM3682 cells either transfected with a miR-211 mimic or scrambled control (D) or treated with TGF-β (2 ng/ml) or DMSO (ctrl) (E). (F and G) Invasion assay of WM3682 melanoma cell grown in adipocyte-conditioned medium, followed by conditioned medium removal (reverse) in the indicated conditions, upon treatment with TGF-β (2 ng/ml; F) or TGF-β inhibitor SB431542 (5 μM; G), each compared with those treated with DMSO. Images are representative; DAPI-stained nuclei are blue. Scale bars, 50 μm. Data in (D) to (G) are means ± SEM from three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.005, t test. (H) Proposed mechanism of adipocyte-mediated regulation of TGF-β signaling in melanoma cells. In proliferative melanoma (melanoma in situ), miR-211 represses the expression of both TGFBR1 (RI) and TGFBR2 (RII), suppressing endogenous TGF-β signaling in melanoma cells and conferring TGF-β resistance. As a result, cells are highly proliferative and weakly metastatic. When adipocytes are present in the melanoma microenvironment, IL-6 and TNF-α released from the adipocytes suppress miR-211 expression. The subsequent expression of TGF-β receptors enhances sensitivity to environmental TGF-β and therefore promotes the melanoma phenotypic switch from the proliferative to the highly invasive state.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/591/eaav6847/DC1

    Fig. S1. Subcutaneous adipocytes are observed in proximity to in situ melanoma.

    Fig. S2. Subcutaneous adipocytes approximate to in situ melanoma correlate with advanced disease.

    Fig. S3. Adipocytes drive melanoma plasticity in a reversible manner by miR-211 repression.

    Fig. S4. Adipocytes decrease miR-211 expression through secretion of IL-6 and TNF-α.

    Fig. S5. miR-211 attenuates TGF-β signaling and reduces melanoma sensitivity to TGF-β.

    Table S1. Characteristics of melanoma cell lines.

    Table S2. Sequence data for oligonucleotides.

    Data file S1. Differentially expressed miRNAs in melanoma upon coculture with adipocytes.

    Data file S2. Gene set enrichment upon miR-211 expression in melanoma.

    Data file S3. Pathway enrichment upon miR-211 expression in melanoma.

    Data file S4. Venn diagram data.

  • The PDF file includes:

    • Fig. S1. Subcutaneous adipocytes are observed in proximity to in situ melanoma.
    • Fig. S2. Subcutaneous adipocytes approximate to in situ melanoma correlate with advanced disease.
    • Fig. S3. Adipocytes drive melanoma plasticity in a reversible manner by miR-211 repression.
    • Fig. S4. Adipocytes decrease miR-211 expression through secretion of IL-6 and TNF-α.
    • Fig. S5. miR-211 attenuates TGF-β signaling and reduces melanoma sensitivity to TGF-β.
    • Table S1. Characteristics of melanoma cell lines.
    • Table S2. Sequence data for oligonucleotides.
    • Legends for data files S1 to S4

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Differentially expressed miRNAs in melanoma upon coculture with adipocytes.
    • Data file S2 (Microsoft Excel format). Gene set enrichment upon miR-211 expression in melanoma.
    • Data file S3 (Microsoft Excel format). Pathway enrichment upon miR-211 expression in melanoma.
    • Data file S4 (Microsoft Excel format). Venn diagram data.

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