CYLD expression is down-regulated in melanoma
To get a first insight into the role of CYLD in malignant melanoma, we evaluated-CYLD expression in six different human melanoma cell lines, as well as in freshly isolated human melanoma cells. We found strong reduction of CYLD mRNA () and protein () levels when compared with normal human epidermal melanocytes (NHEMs). Also, in situ, primary melanomas and melanoma metastases displayed strongly down-regulated or absent CYLD mRNA () and protein (). In contrast, melanocytes in normal epidermis displayed high CYLD expression, as confirmed by costaining for tyrosinase and CYLD and quantification of costaining (). Because mutations and promoter methylation of the CYLD gene could be excluded (unpublished data), we concluded that down-regulation of CYLD likely occurs at the transcriptional level.
Figure 1. Reduced CYLD expression in malignant melanoma. Quantitative RT-PCR (A) and immunoblot analysis (B) showing CYLD expression in six human melanoma cell lines (Mel Im, Mel Juso, Mel Ei, Mel Ju, Mel Ho, and Mel Wei) and freshly isolated primary melanoma cells (more ...)
Snail1 affects transcriptional expression of CYLD in melanoma cells
Analysis of the CYLD
promoter revealed three potential binding sites for the transcriptional repressor Snail1 (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20082044/DC1
). We found that Snail1 messenger RNA (mRNA) was highly increased in melanoma cell lines compared with NHEM, an expression pattern that correlated inversely with CYLD (Fig. S2 A vs. ). In contrast, expression of Snail2 (Snai2), a Snail1 homologue that has been shown to affect expression of several genes important for melanoma progression (21
), in melanoma was similar or lower than in NHEM (Fig. S2 A).
Chromatin immunoprecipitation (ChIP) assays demonstrated Snail1 binding to the CYLD
promoter in primary melanoma cells and the melanoma cell lines Mel Im and Mel Juso, but not in NHEM (). Furthermore, neither Snail2 nor Twist (another E-box binding factor associated with poor prognosis in melanoma [22
]) bound to the CYLD promoter in ChIP assays (). Snail1 recruitment to the CYLD
promoter was observed to the first E-box at position −19 to −14, which resembles the consensus Snail1 binding sequence (CAGGTG; Fig. S1), but not to E-box II (-382 to -377) or III (−429 to −424; unpublished data).
Figure 2. Snail1 inhibits transcriptional expression of CYLD in melanocytic cells. (A) Lysates from NHEMs, primary melanoma cells (MM1 and MM2), and melanoma cell lines (Mel Im, Mel Juso) were examined by ChIP assay using an anti-Snail1, anti-Twist, and anti-Snail2–specific (more ...)
Reporter gene assays using the CYLD promoter (−550 to −1) revealed inducible activity in melanoma cells after transient transfection with an antisense Snail1 construct (as-Snail). Conversely, transient transfection of a Snail1 expression construct completely repressed CYLD promoter activity (). Mutation of the first E-box of the CYLD promoter at position −19 resulted in strong promoter activity in Mel Im cells, which was not affected by transient transfection with asSnail or Snail1 (). In contrast, mutations in E-box II or III of the CYLD promoter did not change the reporter activity (unpublished data).
These data indicate that recruitment of Snail1 to the first E-box binding site located in the CYLD promoter suppresses CYLD promoter activity in malignant melanoma.
To further assess the effect of Snail1 on CYLD expression in melanoma cells, we either stably transfected Mel Im cells with asSnail expression plasmids or depleted Snail1 with specific siRNAs, and found a strong up-regulation of CYLD protein (). Furthermore, transient expression of as-Snail expression plasmids was sufficient to induce CYLD expression in melanoma cell lines and primary human melanoma cells ( and Fig. S2 B). Accordingly, transient transfection of NHEM with a Snail1 expression plasmid strongly inhibited CYLD expression ().
BRAF-mediated ERK activation induced Snail1 and down-regulated CYLD in melanoma cells
Snail1 transcription depends on ERK signaling in squamous cell carcinoma and epithelial cells (15
), and melanoma cells exhibit high ERK activity compared with NHEMs (24
). Interestingly, we found that pharmacological inhibition of the ERK/mitogen-activated protein kinase (MAPK) cascade induced an almost complete loss of Snail1 mRNA expression and significantly increased CYLD mRNA levels in melanoma cells (). In accordance, treatment of NHEM with ERK inhibitors to further reduce basal levels of ERK activity also increased expression of CYLD ().
As BRAF activity acts upstream of ERK, and V600E mutations of BRAF are common in melanoma development (in accordance to our finding that BRAF mutations are present in all melanoma cell lines used in this study [unpublished data]), we tested whether mutated BRAF can modulate CYLD regulation in NHEM. Transfection of NHEM with V600E BRAF resulted in up-regulation of Snail1 and down-regulation of CYLD expression levels ().
Collectively, these data indicate that high Snail1 expression modulated by BRAF-mediated ERK activity is involved in the transcriptional down-regulation of CYLD in melanoma.
Snail1 regulates tumorigenicity in melanoma via CYLD repression
Snail1 has been shown to play an important role in progression of melanoma. To study whether Snail1 facilitates tumorigenicity of melanoma cells via repression of CYLD, the two melanoma asSnail clones () were stably transduced with viral vectors encoding siRNA against CYLD, resulting in complete suppression of CYLD ().
Figure 3. Snail1 regulates tumorigenicity via CYLD repression. (A) Western blot analysis of two Mel Im asSnail clones (clone 1 and clone 2) applying CYLD, Cyclin D1, and N-cadherin antibodies. Stable transduction with viral vectors encoding siRNA against CYLD (siRNA (more ...)
Suppression of Snail1 in Mel Im cells resulted in significantly impaired proliferation (, clone 1; Fig. S3 A, clone 2, available at http://www.jem.org/cgi/content/full/jem.20082044/DC1
) and migration rates (, clone 1; Fig. S3 B, clone 2) compared with control cells. siRNA-mediated depletion of CYLD almost completely rescued the proliferative and migratory potential of Snail1-depleted melanoma cells (, and Fig. S3, A and B).
To assess the relevance of Snail1-mediated tumor progression in vivo, we used a xenograft tumor model. Mel Im asSnail clones formed significantly smaller tumors than Mel Im control cells when injected subcutaneously into nude mice. In contrast, CYLD siRNA reconstituted the diminished growth of Mel Im antisense Snail1 clones (). Histological analysis of tumors formed by Mel Im asSnail clones in nude mice revealed nodular growth (). In contrast, Mel Im asSnail clones transduced with siRNA against CYLD showed more diffuse infiltration of the surrounding tissue ().
These findings indicate that CYLD plays an important role in Snail1-mediated progression of melanoma.
CYLD regulates N-cadherin and Cyclin D1 expression via BCL-3 in melanoma
We have shown in a previous study that BCL-3 associates with the NF-κB p50 or p52 subunits to enhance cell proliferation by activating the Cyclin D1
promoter in mouse keratinocytes (17
). CYLD prevents nuclear accumulation of BCL-3 and hence reduces Cyclin D1 expression and proliferation of keratinocytes.
Next, we examined whether a similar mechanism operates in human melanoma cells. In contrast to NHEM, BCL-3 was primarily found in the nucleus of the melanoma cell lines (Fig. S4 A, available at http://www.jem.org/cgi/content/full/jem.20082044/DC1
). Transduction with lentiviral vectors expressing CYLD prevented nuclear translocation of BCL-3 (Figs. S4, B and C), whereas a catalytically inactive mutant of CYLD (CYLD C/S) still binding to BCL-3 was unable to prevent nuclear translocation (Fig. S4, C and D).
To analyze whether CYLD is also regulating Cyclin D1 expression in melanoma cell lines, Mel Im and Mel Juso cells were transduced with viral vectors carrying CYLD. Clones expressing CYLD at similar levels as NHEMs were used as demonstrated by Western blot analysis (). In agreement with the mechanism in keratinocytes (17
), CYLD markedly reduced expression of Cyclin D1 (), cyclin D1
promoter activity (), and BCL-3 recruitment to the cyclin D1 promoter in a complex with p50 or p52 (, left) as compared with cells transduced with viral vectors carrying a catalytically inactive mutant of CYLD (C/S-CYLD), a GFP expression cassette or noninfected cells.
Figure 4. CYLD regulates N-cadherin and Cyclin D1 expression via BCL-3 in melanoma. (A) Western blot analysis revealing CYLD expression in NHEMs and Mel Im and Mel Juso cells stably transduced with CYLD, but not in melanoma cells stably transduced with GFP. Actin (more ...)
Because melanoma cells with BRAF V600E mutation exhibit an increased production of the immunosuppressive IL-10 (25
), and the production of IL-10 is reduced in BCL-3–null cells (26
), we further analyzed BCL-3 binding to the IL-10 promoter. CYLD, but not C/S-CYLD, abrogated BCL-3 binding of IL-10 promoter, confirming the important role of CYLD in the regulation of BCL-3 (Fig. S4 E).
In addition to uncontrolled proliferation, malignant melanoma is characterized by early invasiveness and metastasis. One of the hallmarks of the increased migratory and invasive potential of malignant melanoma cells is the expression of N-cadherin (2
). Interestingly, the N-cadherin
gene contains NF-κB binding sites in its promoter region (27
). ChIP analysis revealed DNA-bound BCL-3 in a complex with p50 or p52 in control cells, and CYLD C/S– or GFP-expressing cells, whereas CYLD-expressing cells showed no BCL-3 recruitment to the NF-κB binding sites of the N-cadherin
promoter (, right).
In line with these findings, expression of CYLD in melanoma cells strongly reduced N-cadherin promoter activity () and protein levels (), whereas no effects on Snail1, Snail2, Twist, or E-cadherin expression () were found compared with GFP- or CYLD C/S–transduced controls, respectively. In agreement, down-regulation of CYLD in asSnail clones 1 and 2 resulted in up-regulation of Cyclin D1 and N-cadherin ().
Depletion of BCL-3 expression in melanoma cells using siRNA resulted in reduced expression of both N-cadherin and Cyclin D1 without changes in Snail1 expression levels (). Furthermore, siRNA against Snail1, which up-regulated CYLD expression (), reduced nuclear BCL-3 levels, confirming that reexpression of CYLD blocks nuclear translocation of BCL-3 in melanoma ().
These results suggest that BCL-3–induced cyclin D1 and N-cadherin expression is blocked by the expression of CYLD.
CYLD regulates proliferation and N-cadherin–mediated migration and invasion in melanoma
To investigate the functional role of the suppressive effect of CYLD on Cyclin D1 and N-cadherin, we used the two different melanoma cell lines (Mel Im and Mel Juso) stably expressing CYLD (). CYLD expression caused diminished proliferation in Mel Im cells () and Mel Juso cells (Fig. S5 A, available at http://www.jem.org/cgi/content/full/jem.20082044/DC1
), as well as a decrease in colony growth in soft agar in both cell lines ( and Fig. S5 B) compared with control, GFP, or CYLD C/S–infected cells.
Figure 5. CYLD regulates proliferation and N-cadherin–mediated migration and invasion. (A) Proliferation of Mel Im cells expressing CYLD, CYLD C/S, or GFP versus the parental Mel Im cell line (Control) after 72 h. Data are given as mean ± SEM. *, (more ...)
Analysis of the motile and invasive phenotype of CYLD-expressing melanoma cells revealed that CYLD expression resulted in diminished migration and invasion rates as assessed in time-lapse scratch assays ( and Fig. S5 C), spheroid migration assays ( and Fig. S5 D), and Boyden chamber assays ( and Fig. S5 E).
In contrast, forced expression of N-cadherin reversed the CYLD-mediated reduction in melanoma cell migration ( and Fig. S5 F), and depletion of BCL-3 expression resulted in diminished migration rates ( and Fig. S5 G).
These data indicate that CYLD regulates proliferation, as well as migration and invasion, of melanoma by controlling N-cadherin expression.
CYLD inhibits proliferation and metastasis of melanoma cells in vivo
To test the effect of CYLD on tumor growth in vivo, we used a murine xenograft model. Mel Im cells stably expressing CYLD were injected subcutaneously into nude mice, revealing significantly impaired growth compared with cells stably expressing GFP or noninfected cells (). Similar results were obtained with Mel Juso cells (Fig. S6 A, available at http://www.jem.org/cgi/content/full/jem.20082044/DC1
). 14 d after injection, none of the mice from the CYLD group, but 6 out of 10 animals from the GFP and noninfected control groups, developed tumors. After 4 wk, 4 mice from the CYLD group also developed tumors; however, these tumors were significantly smaller (4.3 ± 2.8 vs. 28.6 ± 14.7 mm3
[GFP] and 28.5 ± 10.7 mm3
Figure 6. CYLD inhibits proliferation and metastasis of melanoma cells in vivo. (A) Growth kinetic of tumors formed by Mel Im control cells (Control) or cells transduced with viral vectors carrying CYLD or GFP after subcutaneous implantation into nude mice (10 (more ...)
In addition, melanoma cells were injected i.v. into nude mice to assess in vivo metastasis. MIA serum levels are an established marker to monitor melanoma metastasis (28
), and therefore, an ELISA selectively detecting human MIA was applied to monitor the serum content of MIA in this xenograft model. 4 wk after injection, animals receiving CYLD-expressing melanoma cells had significantly lower MIA serum levels (1.1 ± 0.4 ng/ml) than animals injected with GFP-transduced (3.8 ± 0.8 ng/ml) or nontransduced (3.8 ± 1.6 ng/ml) cells ( and Fig. S6 B).
We performed immunohistochemical analysis sections using MART 1 and MIA, two tumor-associated antigens, to detect lung metastasis. Mice receiving Mel Im cells stably transduced with CYLD showed almost no MART 1 () or MIA (not depicted) staining compared with mice injected with control cells, indicating that they had significantly less metastatic deposits ( and Fig. S6 C).
In summary, these data show that reduced CYLD expression induces tumorigenicity of melanoma, and that reexpression of CYLD reduces their tumor growth and metastasis in vivo.
Clinical relevance of Snail1-induced CYLD repression
To analyze the clinical relevance of reduced CYLD expression in melanoma, we performed immunohistochemical analysis of a tissue microarray consisting of 88 primary human melanomas () and found a significant correlation between loss of CYLD expression and Clark level/tumor invasiveness and tumor thickness (). Moreover, loss of CYLD expression inversely correlated with overall () and progression-free survival (). In contrast, patients with primary tumors expressing CYLD developed no tractable metastasis in the time period analyzed.
Figure 7. CYLD expression in melanoma has prognostic implication. Kaplan-Meier curves for overall survival (A) and progression-free survival (B) in melanoma patients with a positive immunosignal for CYLD (CYLD positive) or without detectable CYLD protein expression (more ...)
CYLD and Snail1 immunoreactivity in relation to clinico-pathological characteristics
Notably, and in accordance with the Snail1-CYLD connection described here, expression of Snail1 was inversely correlated to CYLD expression in primary tumors (P < 0.001; ). In compliance, Snail1 expression also correlated with tumor thickness () and overall () and progression-free survival ().
These clinical findings support a link between the known tumor promoter Snail1 and its transcriptional target CYLD, indicating an important role for Snail1 to suppress CYLD during progression of melanoma and for CYLD to suppress melanoma cell proliferation and invasion.