Epithelial-mesenchymal-transition-like and TGFβ pathways associated with autochthonous inflammatory melanoma development in mice

Abstract

We compared gene expression signatures of aggressive amelanotic (Amela) melanomas with those of slowly growing pigmented melanomas (Mela), identifying pathways potentially responsible for the aggressive Amela phenotype. Both tumors develop in mice upon conditional deletion in melanocytes of Ink4a/Arf tumor suppressor genes with concomitant expression of oncogene H-Ras(G12V) and a known tumor antigen. We previously showed that only the aggressive Amela tumors were highly infiltrated by leukocytes concomitant with local and systemic inflammation. We report that Amela tumors present a pattern of de-differentiation with reduced expression of genes involved in pigmentation. This correlates with reduced and enhanced expression, respectively, of microphthalmia-associated (Mitf) and Pou3f2/Brn-2 transcription factors. The reduced expression of Mitf-controlled melanocyte differentiation antigens also observed in some human cutaneous melanoma has important implications for immunotherapy protocols that generally target such antigens. Induced Amela tumors also express Epithelial-Mesenchymal-Transition (EMT)-like and TGFβ-pathway signatures. These are correlated with constitutive Smad3 signaling in Amela tumors and melanoma cell lines. Signatures of infiltrating leukocytes and some chemokines such as chemotactic cytokine ligand 2 (Ccl2) that contribute to leukocyte recruitment further characterize Amela tumors. Inhibition of the mitogen-activated protein kinase (MAPK) activation pathway in Amela tumor lines leads to reduced expression of EMT hallmark genes and inhibits both proinflammatory cytokine Ccl2 gene expression and Ccl2 production by the melanoma cells. These results indicate a link between EMT-like processes and alterations of immune functions, both being controlled by the MAPK pathway. They further suggest that targeting the MAPK pathway within tumor cells will impact tumor-intrinsic oncogenic properties as well as the nature of the tumor microenvironment.

Figure 1. Differential expression in Amela and Mela tumors of genes involved in pigmentation, differentiation and development of melanocytes.

A. Microarray data as log2 for Amela and Mela tumors are shown in arbitrary units (a.u.). For these genes, ratio of gene expression as log2 Amela/Mela is < -1 (a) or between -1 and 0 (b) with p values < 0.05; for Pou3f2 (c), ratio of gene expression as log2 Amela/Mela is > 1 with p value < 0.05. B–C. Validation of expression of four genes (from A) by QRT-PCR in Amela and Mela tumors ex vivo (B) and of corresponding melanoma lines cultured in vitro (C). D–E. Relative expression of transcripts for Mitf (D) and for Brn2/Pou3f2 (E) in induced Mela and Amela tumors ex vivo. Amela tumors are represented by white dotted bars and Mela tumors by black dotted bars. For ex vivo analysis (B, D, E) values were normalized to those for skins of control mice, 6 samples of each tumor and 4 skin samples were analyzed. For in vitro analysis (C), 3 different cDNA preparations from two Mela and 8 Amela tumor lines were used and values were normalized to those for B16F10 cells. ***p value < 0.001; **p value < 0.01; *p value < 0.05 (see methods).

Figure 2. EMT and TGFβ pathway signatures in Amela melanomas. A.

Representative gene set enrichment analysis (GSEA) plots of EMT (right graph) and TGFβ pathway (left graph) gene signatures. Each plot is divided into two sections. The first section (class A) shows results for gene sets that have a positive enrichment score (gene sets that show enrichment at the top of the ranked list here associated with Amela samples). The second section (class B) shows the results for gene sets that have a negative enrichment score (gene sets that show enrichment at the bottom of the ranked list here associated with Mela samples). Data provided as in Fig. S2. B. QRT-PCR analysis for expression of transcripts encoding mesenchymal and epithelial markers in Amela (white dotted bars) and Mela (black dotted bars) tumors. Values were normalized to those for skin from control mice. Data are represented as mean ± s.e.m of three independent experiments in which 7 samples of Amela and 7 samples of Mela tumors were analyzed. **p value < 0.01; *p value < 0.05.

Figure 3. Analysis of the TGFβ pathway in melanoma lines and tumors. A.

Supernatants from Amela cell lines incubated in serum-free DMEM were either acid treated (acid) or not (no treatment) and were tested for TGFβ content using reporter line MFBF11 (see Methods). Bars represent means ± s.d. of triplicate wells in one representative experiment. Serum-free DMEM (no TGFβ) was used for baseline measurement. B. Flow cytometry analysis of GFP expression in SBE-GFP-transduced Amela and B16F10 cell lines preincubated in serum-free DMEM without addition (red lines) or in the presence of TGFβ (blue lines). Non-transduced Amela cell lines were used as control (gray filled). C. Comparasion of the expression of 3 genes associated with EMT by Amela^C and Amela^I cell lines by QRT-PCR (see text). Results are represented as fold change in relative expression where the value for Amela^C is set to 1 for each gene. Data are represented as mean ± s.e.m of two independent experiments in which 4 different lines of each type were tested. D-E. Flow cytometry analysis of GFP expression in SBE-GFP-transduced Amela^C cell lines as in (B). The effect of various inhibitors was assessed on Amela^C cells during the incubation in serum-free DMEM without (-) or with addition (+) of TGFβ. GFP expression in one Amela^C line before and after treatment with various inhibitors in the absence of TGFβ is shown in (D). In (E)results show fold change of mean GFP fluorescence intensity in Amela^C without (panel at left) or with (panel at right) addition of TGFβ. The value for Amela^I in the absence of TGFβ is set to 1. Data are represented as mean ± s.e.m of four independent experiments. ***p value < 0.001; **p value < 0.01; *p value < 0.05.

Figure 4. Effect of MAPK pathway inhibitors on the expression of EMT hallmark genes in Amela tumor lines.

QRT-PCR analysis for expression of 6 transcripts encoding mesenchymal and epithelial markers in Amela tumor cells in the absence (value set to 1) or presence of inhibitors, as indicated. Data are represented as mean ± s.e.m of three independent experiments in which 5 samples were analyzed. ***p value < 0.001; **p value < 0.01; *p value < 0.05.

Figure 5. Analysis of phosphorylated Smad3 and JNK in melanoma tumors.

Sections of Amela and Mela tumors were analyzed by immunohistology after staining with anti-CD45 mAb (green) and with anti-Phospho-Smad3L (pSmad3L) (A) or with anti-Phospho-JNK (pJNK) (B) (red). Sytox blue stains nuclei (white). Bars correspond to 50 µm in the 4 quadrant-figures and to 20 µm in the magnifications of the highlighted fields (labeled 1 and 2). In A, the brightfield image is superimposed on the “merge” image. Images are representative of 3 tumors of each type. Immunofluorescence was quantified using NIH ImageJ software for the determination of relative densities of expression of given markers within fixed section areas (fraction area). Values were, respectively (A) 1.95 ± 0.73 for Mela and 13.78 ± 1.55 for Amela tumor Phospho-Smad3L staining (unpaired t test p < 0.0001) and (B) 5.39 ± 1.05 for Mela and 26.36 ± 3.26 for Amela Phospho-JNK staining (unpaired t test p < 0.0005).

Figure 6. Specific expression of Ccl2 in Amela tumors is controlled by Ras signalling pathways.

(A-B) QRT-PCR analysis of Ccl2 transcript expression in induced Mela and Amela primary tumors (A) and in the corresponding cell lines in vitro (B). (C) Concentration of secreted Ccl2 in the supernatant of 24 h cultures of Amela and Mela cell lines as measured by ELISA. (D) Expression of Ccl2 transcript by QRT-PCR and (E) concentration of secreted protein by ELISA, in the absence (value set to 1) or presence of inhibitors in 24 h cultures, as indicated. (A–C) Amela tumors are represented by white dotted bars and Mela tumors by black dotted bars. For primary tumor analysis (A) 6 samples of each tumor were analyzed. For in vitro analysis (B), values were normalized to those for B16F10 cells. In (C), supernatants from 24 h cell cultures were tested and values are expressed as ng/ml. In D–E, culture conditions were as described in Methods. Experiments involved 10 Amela lines in (D) and 4 in (E). ****p value < 0.0001; ***p value < 0.001; **p value < 0.01; *p value < 0.05.