In melanoma and other cancers, invasion, epithelial-to-mesenchymal transition, metastasis and cancer stem cell maintenance are regulated by transcription factors including the Snail family. Slug (Snail2) protein generally supports migration and apoptosis resistance. However, its role in melanoma is not completely understood. The present study investigated the transcriptional regulation of the
Hedgehog (HH) signaling is a developmentally conserved pathway in numerous embryonic tissues and has been shown to be dysregulated in multiple cancers (
The HH pathway has been shown to be essential for the oncogenic properties of melanoma (
The transcription factor Slug, the protein product of the
In melanoma,
Despite these findings, the precise mechanisms of Slug expression and its role in EMT remain to be elucidated in melanoma. Gupta
The present study used eight melanoma cell lines (listed in
GANT61 (stock prepared in DMSO) and cyclopamine (stock prepared by dissolving in ethanol) were purchased from Selleck Chemicals LLC. The chemicals were applied to cells as indicated in the appropriate figures for 20 h before cell harvesting if not stated otherwise. The addition of 20 µM GANT61 for 20 h was used after the optimization of both the concentration and time to follow the changes in expression of GLI-dependent genes, when no signs of apoptosis had been yet detected in cells.
To obtain whole-cell extracts for immunoblotting analysis, cells were lysed in RIPA buffer (1% NP-40, 150 mM NaCl, 5 mM EDTA, 0.5% sodium deoxycholate, 50 mM Tris-HCl pH 7.5 and 0.1% SDS) with the addition of the protease and phosphatase inhibitors aprotinin, pepstatin and leupeptin at 1 mg/ml each. cOmplete (Roche Diagnostics) was added as recommended by the supplier. Then, 1 mM phenylmethylsulfonyl fluoride (MilliporeSigma) and phosphatase inhibitor PhosSTOP (Roche Diagnostics) were added. Equal amounts of protein (30 µg; concentration determined by Bradford's assay) were loaded on 10–12% SDS-polyacrylamide gels, separated by electrophoresis and transferred onto PVDF membranes. Blots were blocked in 5% non-fat dry milk Blotto (cat. no. sc-2325, Santa Cruz Biotechnology, Inc.), and incubated with 1:1,000 diluted primary antibodies for 2 h, washed, and then incubated at room temperature for 1 h with 1:4,000 diluted secondary anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (cat. no. sc-2055 or cat. no. sc-2030; Santa Cruz Biotechnology, Inc.). Chemiluminescent detection was used. For western blotting shown in
The 12×GLI-TK-Luc plasmid was obtained from Professor R Toftgard, Karolinska Institute, Sweden. pGL3-PTCH1 was kindly donated by Professor Aberger, University of Salzburg, Austria. The
Transient cell transfections of the promoter reporters were performed using 12-well plates at 37°C, seeded and incubated for 24 h before transfections, and the transfection reagent Mirus
Total RNA was isolated using TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the supplier´s instructions (3×105 cells per 30 mm well). Then 2 µg of RNA was reverse transcribed using SuperScript IV reverse transcriptase (Thermo Fisher Scientific, Inc.), and qPCR was performed using a TaqMan QuantiTect Probe PCR kit (Qiagen GmbH) on a ViiA7 Real-Time PCR system (Thermo Fisher Scientific, Inc.) following the manufacturer's instructions (cycling: 30 sec at 94°C and 1 min at 60°C). Data analysis was performed by QuantStudio 6 Software (Thermo Fisher Scientific, Inc.). Concurrent results were obtained in three independent experiments with the following PCR primers and probe for
501mel cell cultures were transfected using Mirus
For immunofluorescence, four cell lines (501mel, Hbl, SK-MEL-5 and SK-MEL-28) were seeded into 8-well Lab-Tek II Chamber Slides (Thermo Fisher Scientific, Inc.). After 48 h, 20 µM GANT61 was added for 20 h at 37°C. Vehicle (DMSO) alone was added to the controls. The cells were fixed with 3% paraformaldehyde at room temperature for 10 min, washed, permeabilized with 0.1% Triton X-100, and blocked with 5% goat serum. Slides were then stained with Slug primary antibody (1:1,000; cat. no. sc-166476; Santa Cruz Biotechnology, Inc.) at room temperature for 2 h followed by 1:1,000 secondary anti-mouse fluorescein-coupled antibody (cat. no. FI-2000-1.5, Vector Laboratories, Inc.) and mounted in medium with DAPI to stain nuclei. The visualization was performed using an Olympus IX70 microscope with cellSens software V2.2 (Olympus Corporation).
Formalin-fixed paraffin-embedded tissues (skin, nevus and melanoma) were retrieved from the archive of the Department of Pathology and Molecular Medicine, Second Faculty of Medicine, Charles University, University Hospital Motol, Prague. At least four samples of each tissue were processed and similar results were obtained. Deparaffinized, rehydrated in descending ethanol series, and blocked (with 3% H2O2) sections were stained with 1:1,000 primary antibodies. Immunohistochemistry for MITF was performed with the primary antibody MITF (cat. no. D5; cat. no. NBP2451590, Thermo Fisher Scientific, Inc.). GLI2 was stained with antibody cat. no. GTX46056 (GeneTex, Inc.) and Slug with anti-Slug (A-7) sc-166476 antibody (Santa Cruz Biotechnology, Inc.). Detection was performed using an EnVision+ avidin-biotin detection system (Dako; Agilent Technologies, Inc.). Each tissue was examined by two pathologists. Tissues were scored on a scale of 0 to 4 based on the combined extent and intensity of staining. The final score represented the predominant staining pattern of both combined parameters. Section fields were imaged using a BX51 microscope (Olympus Corporation) equipped with a PROMICAM 3-3CP 3.1 camera and QuickPHOTO CAMERA 3.2 software (Olympus Corporation).
Statistical significance (P-values) was assessed using a two-tailed Student's t test and Mann-Whitney test. Standard error of the mean values are depicted in graphs as bars within each column in the reporters and RT-qPCR. Data not significant (P>0.05) were not labeled, values of 0.01 <P<0.05 are marked by an asterisk, and values with P<0.01 are marked by two asterisks. SigmaPlot software V10.0 (Systat Software Inc.) and GraphPad Prism v.8.4.3 software (Dotmatics) were used to perform statistical analysis. Statistical analysis was verified using one-way ANOVA followed by post hoc tests as specified in figure legends. P<0.05 was considered to indicate a statistically significant difference.
Snail1 and Snail2 (Slug) are among the main players in the tumorigenic program of EMT (
To further investigate this hypothesis, Slug protein expression was examined in melanoma cell lines treated with GANT61, a potent and specific pan-GLI transcriptional inhibitor. It was found that GANT61 decreased Slug protein in all eight melanoma cell lines investigated as well as in normal melanocytes (
Since the transcriptional inhibitor of GLI factors GANT61 downregulated Slug protein expression, the activity of the
Next, to test whether the SLUG promoter-reporter is directly activated by cotransfected expression vectors for GLI factors and inhibited by GANT61, it was first investigated whether the proximal promoter activity decreased after the addition of HH pathway inhibitors. Indeed, both GANT61 and cyclopamine lowered the activity in all three melanoma cell lines tested (
To further evaluate the transcriptional regulation of Slug by HH/GLI, the present study examined the effect of GANT61 on Slug RNA levels using real-time PCR. RT-PCR was performed first, followed by qPCR. In all eight melanoma cell lines tested, 20 µM GANT61 significantly (P<0.01) lowered the mRNA level of Slug. This indicates that the positive effect of GLI factors on endogenous
The proximal
The
To corroborate these results, doxycycline-regulatable melanoma cell lines, in which MITF could be downregulated by inducible expression of shRNA directed at MITF were used (
To further investigate the relationship between MITF and Slug and GLI2 vs. Slug protein expression in human samples, parallel sections of skin, nevus and melanoma metastasis were compared by immunohistochemical staining (
The present study described the essential role of HH signaling and the transcription factor GLI2 in the transcription of the
Previously, the essential role for HH signaling in melanoma has been demonstrated to occur mainly through the transcription factor GLI1 (
It has been demonstrated that the oncogene
HH pathway and GLI factors are highly oncogenic and known to substantially contribute to the maintenance of CSC. In addition, GLIs are observed to be associated with EMT in various types of cancer (
In melanoma, therapy is predominantly focused on targeting the mutated driver oncogenes BRAF and NRAS and/or kinases in the downstream MAPK signaling pathway. Unfortunately, therapies for advanced melanoma with low molecular weight inhibitors targeting these proteins result in acquired resistance. Despite advances in using a combination of drugs, the concept of targeting only the MAPK route remains questionable. As there are multiple mechanisms responsible for resistance to the inhibition of MAPK signaling in melanoma (
The authors thank Professor F. Aberger (University of Salzburg) for providing the PATCHED promoter plasmid and Professor R. Toftgard (Karolinska Institute) for the 12×GLI reporter plasmid.
All data generated or analyzed during this study are included in this published article.
JV and PH conceived the project. JV and PH confirm the authenticity of all the raw data. PH, JR, KK, LO, JV and PZ performed the experiments. JB and JV Jr. performed the immunohistochemical experiments. PH and KK performed statistical analyses. PH and KK prepared the figures. PH and JV wrote the manuscript. All authors read and approved the manuscript.
Immunohistochemical analysis of paraffin sections of human tissues was approved by the Ethical Committee of the University Hospital Motol, Prague (approval no. EK-36/20). The study was carried out in accordance with the Declaration of Helsinki.
Not applicable.
The authors declare that they have no competing interests.
snail family zinc finger 2
snail family zinc finger 1
epithelial-to-mesenchymal transition
aldehyde dehydrogenase 1 family member A1
Krüppel-like transcription factor 4
GLI family zinc finger
cancer stem cells
Hedgehog signaling pathway
melanoma-associated transcription factor
Distribution of GLI-binding sites in the
Promoter-reporter analysis of the
GANT61 decreases Slug RNA levels. mRNA levels of Slug were assessed by RT-qPCR quantification. A total of eight cell lines were treated with 20 µM GANT61 for 20 h, and total RNA was isolated. After RT, RT mixes were analyzed by qPCR. The relative mRNA levels of Slug were normalized to those of β-actin. Values are presented as the mean ± standard error of the mean. Three independent experiments were performed, each in triplicate. Similar results were obtained in all experiments, and one is depicted. The nontreated control (vehicle) was set as 1.0. All decreases in Slug RNA were statistically significant (**P<0.01) by Student's t test. RT-qPCR, reverse transcription-quantitative PCR.
Binding of GLI factors to the proximal
MITF is an imperfect activator of Slug in 501mel melanoma cells. (A) Left,
Slug and GLI2 expression correlate in serial sections of normal skin, nevus, and melanoma. Immunohistochemical staining was performed with antibodies against GLI2, Slug and MITF. For staining scores, see Results section (magnification, 400×). GLI, GLI family zinc finger; MITF, melanoma-associated transcription factor.