The MNT-1 human pigmented melanoma cell line, kindly gifted by Profs. Michael S. Marks (Children's Hospital of Philadelphia and University of Pennsylvania, PA) and Cheah Shiau Chuen (UCSI University, Kuala Lumpur, Malaysia), was cultured with Dulbecco's Modified Eagle Medium (Nissui Pharmaceutical, Tokyo, Japan) that was supplemented with 7% (v/v) FBS, 10% (v/v) AIM-V Medium liquid (Thermo Fisher Scientific, Inc. Waltham, MA), 100 U/ml penicillin G, 100 mg/l kanamycin, 2.25 g/l NaHCO₃, and 600 mg/l L-glutamine at 37°C in a humidified incubator with 5% CO₂. The SK-MEL-28 human amelanotic melanoma (RIKEN BioResource Center, Tsukuba, Japan), WM266-4 (American Type Culture Collection, Manassas, VA), 293T human embryonic kidney (RIKEN BioResource Research Center), and HT1080 human fibrosarcoma (Japanese Collection of Research Bioresources Cell Bank, Osaka, Japan) cell lines were cultured in Dulbecco's Modified Eagle Medium that was supplemented with 7% (v/v) FBS, 100 U/ml penicillin G, 100 mg/l kanamycin, 2.25 g/l NaHCO₃, and 600 mg/l L-glutamine at 37°C in a humidified incubator with 5% CO₂.

Arbutin (Merck KGaA, Darmstadt, Germany) and α-melanocyte-stimulating hormone (α-MSH; Peptide Institute, Inc., Osaka, Japan) were dissolved in sterilized water. 3-isobutyl-1-methylxanthine (IBMX; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was dissolved in dimethyl sulfoxide (DMSO).

Cell pellets were dissolved with 10% (v/v) DMSO that contained 1 N NaOH at 70°C for 1 h. The melanin content was quantified by measuring the absorbance at 405 nm. Each absorbance value was normalized by the amount of total protein.

The enzymatic activity of cellular TYR was quantified using the Tyrosinase Activity Assay Kit (Abcam # ab252899; Cambridge, UK) according to the manufacturer's instructions.

The in vitro VM assay was conducted as described (29–31). Initially, 96-well plates were coated with 40 µl/well of Matrigel^® Growth Factor Reduced (Corning, Corning, NY) and incubated for 30 min at 37°C. Cells were suspended and added to the Matrigel-coated wells at 2.0×10⁴ cells/well and cultured at 37°C in a humidified incubator with 5% CO₂. In each well, images of five independent, randomly selected fields were captured using phase-contrast microscopy (Leica DMi1, Leica, Wetzlar, Germany), and the number of tubes was counted. A tube was defined as an area that was surrounded by cells.

Cells were seeded at 2.0×10³ cells/well in the presence of vehicle, IBMX, or α-MSH in 96-well plates and cultured for 48 h. Thiazolyl blue tetrazolium bromide was added to each well, after which the cells were cultured for 4 h at 37°C. The media was removed, and the MTT formazan product was dissolved in 100 µl DMSO. The absorbance at 570 nm was measured to quantify the number of living cells.

Knockout (KO) of TYR was performed using the CRISPR/Cas9 system as described (31,32). We used the D10A Cas9 mutant to avoid off-target effects (called the Nickase system). Thus, we designed 2 nearby targets in exon 1 of TYR; the sequences of the oligonucleotides for generating the guide RNAs were as follows: target 1, 5′-CACCGGGCTCTAGGGAAATGGCCAG-3′ (forward) and 5′-AAACCTGGCCATTTCCCTAGAGCCC-3′ (reverse); and target 2, 5′-CACCGTGTCTCCTCTAAGAACCTGA-3′ (forward) and 5′-AAACTCAGGTTCTTAGAGGAGACAC-3′ (reverse). Each pair of oligonucleotides was annealed and inserted into the BbsI restriction site of pSpCas9n(BB)-2A-Puro (PX462) V2.0 (gifted by Feng Zhang, Addgene, Cambridge, MA). MNT-1 and SK-MEL-28 cells were cotransfected with these plasmids using Lipofectamine 3000™ (Thermo Fisher Scientific, Inc.) and then treated with 1.25 µg/ml puromycin dihydrochloride (Merck KGaA) to select transfectants. Clonal TYR-KO cells were established by limiting dilution method.

TYR cDNA was amplified from the pcDNA4(TO)-tyrosinase plasmid (33), a kind gift of Prof. Takafumi Hasegawa (Tohoku University, Sendai, Japan), by polymerase chain reaction (PCR) using the following primers: 5′-TTTTCTCGAGATGCTCCTGGCTGTTTTGTACTGC-3′ (forward) and 5′-TTTTGCGGCCGCTTATAAATGGCTCTGATACAAGCTGTGG-3′ (reverse). To avoid recognition by Cas9, we constructed Cas9-resistant TYR cDNA by overlap extension PCR with the following primers: 5′-GCGTGAGCAGCAAAAATCTCATGGAAAAGGAATGCTGTCCACCGTG-3′ (forward) and 5′-AAGCCCGTGGAAAGTGTCCGGCGCTGGTCTGGAAACTCCACAGCAG-3′ (reverse). The T373K point mutation was generated by overlap extension PCR with the following primers: 5′-AATGGAAAAATGTCCCAGGTACAGGGATCTG-3′ (forward) and 5′-TTTGGATGAAATAAAGAAATCACCATTTCTG-3′ (reverse). The resulting amplicons were inserted into the XhoI/NotI restriction site of CSII-CMV-MCS-IRES2-Bsd (RIKEN BioResource Center). The CSII-CMV-MCS-IRES2-Bsd-GFP plasmid (34) was used as a control.

293T cells were transfected with these plasmids using Lentivirus High-Titer Packaging Mix (Takara Bio Inc.) and cultured for 6 h. The cells were washed with phosphate-buffered saline and cultured with fresh medium for 42 h to facilitate the production of virus particles. TYR-KO MNT-1 and WM266-4 cells were then treated with the lentivirus-containing conditioned media. After infection, cells were selected with 12.5 µg/ml blasticidin S (FUJIFILM Wako Pure Chemical Corporation).

Western blot was performed as described (31,35). Cells were cultured and lysed in lysis buffer [50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% (w/v) SDS, 1% (v/v) Triton X-100, 1% (w/v) sodium deoxycholate, and 1 mM phenylmethylsulfonyl fluoride] with PhosSTOP phosphatase inhibitor cocktail (Merck KGaA) on ice with sonication. The lysate was centrifuged at 15,300 × g for 10 min, and the supernatant was collected. The amount of protein in each cell lysate was measured by Coomassie Brilliant Blue G-250 staining (Bio-Rad Laboratories, Inc., Hercules, CA).

Loading buffer [350 mM Tris-HCl pH 6.8, 30% (v/v) glycerol, 0.012% (w/v) bromophenol blue, 6% (w/v) SDS, and 30% (v/v) 2-mercaptoethanol] was added to each lysate and boiled for 3 min. The samples were electrophoresed on 9% SDS-polyacrylamide gels, after which the proteins were transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk at room temperature for 30 min and immunoblotted with monoclonal anti-TYR (Abcam # ab170905), monoclonal anti-α-tubulin (Merck KGaA #T5168), monoclonal anti-CREB (Cell Signaling Technology #9197; Danvers, MA), and anti-phospho-CREB (Cell Signaling Technology #9191) at room temperature for 1 h. HRP-linked anti-rabbit IgG (Cytiva #NA934; Marlborough, MA) and HRP-linked anti-mouse IgG (Cytiva #NA931) were added to the membranes for 1 h at room temperature. Signals were detected by enhanced chemiluminescence using Western Lightning Plus-ECL (PerkinElmer, Inc., Waltham, MA) or Immobilon Western Chemiluminescent HRP substrate (Merck KGaA) and exposed to RX-U films (FUJIFILM, Tokyo, Japan) in a dark room.

Differences between 2 groups were analyzed by two-tailed student's t-test (unpaired). Datasets with 3 groups or over were analyzed using one-way ANOVA with Tukey's test using SPSS (version 27; IBM, Armonk, NY). The results were expressed as mean ± SD. P<0.05 was considered to indicate a statistically significant difference.

To determine the significance of cAMP/CREB/TYR signaling in VM, we treated MNT-1 cells with the cAMP signaling activator IBMX and confirmed that IBMX induces phosphorylation of CREB and upregulates TYR (Fig. 1A and B). IBMX also inhibited VM in MNT-1 cells (Fig. 1C). Further, α-MSH, a potent activator of cAMP signaling, increased CREB phosphorylation and TYR levels (Fig. 1A and B). Consistent with this result, α-MSH impeded VM in MNT-1 cells (Fig. 1D).

Cotreatment with IBMX and α-MSH enhanced the expression of TYR and inhibited VM in MNT-1 cells (Fig. 1B and E). IBMX and α-MSH did not affect cell viability individually or in combination (Fig. S1), confirming that their suppressive activities on VM were not attributed to cell death. These data suggest that IBMX and α-MSH inhibit VM, consistent with the activation of the cAMP/CREB/TYR axis.

Because α-MSH and IBMX upregulated TYR (Fig. 1B), we examined the function of TYR in VM. Arbutin is a well-known TYR inhibitor and has inhibitory effects on melanin synthesis (36), and we confirmed the reduction of melanin content in arbutin-treated MNT-1 cells (Fig. 2A). By contrast, arbutin promoted VM (Fig. 2B and C), prompting us to study the effects of arbutin on VM in non-melanoma cell lines. HT1080 is a TYR-non-expressing tumor cell line (Fig. S2A). By VM assay, arbutin did not increase tube numbers in HT1080 cells (Fig. S2B), indicating that arbutin suppresses VM by inhibiting TYR in tumor cell lines.

To verify the function of TYR in VM, we established a TYR-KO MNT-1 cell line using the CRISPR/Cas9 system (Fig. 3A). As expected, the enzymatic activity of TYR decreased significantly, and thus, melanin content was diminished in TYR-KO MNT-1 cells (Fig. S3A and SB). Consistent with the results after treatment with arbutin, VM was promoted in TYR-KO MNT-1 cells (Fig. 3B). Given that amelanotic melanomas also express TYR endogenously, we examined whether TYR has suppressive activity against VM even in amelanotic melanoma cells. To test this, we deleted TYR in SK-MEL-28 human amelanotic melanoma cells by CRISPR/Cas9 and confirmed its enzymatic activity (Figs. 3C and S4A). As shown in Fig. 3D, depletion of TYR promoted VM in SK-MEL-28, as well as pigmented MNT-1 cells. We also confirmed that overexpression of TYR increases its enzymatic activity and attenuates VM in WM266-4 human amelanotic melanoma cells (Fig. S4B-SD). In addition, KO of TYR attenuated inhibitory effect of α-MSH on VM in MNT-1 cells (Figs. 1D and S5). These results demonstrate that TYR suppresses VM in pigmented and amelanotic melanoma cells.

Human TYR often carries mutations, some of which cause albinism (37). The T373K mutation is frequently observed in albinos, attenuating the enzymatic activity of TYR (38). Thus, we re-expressed wild-type (wt) or T373K TYR in TYR-KO MNT-1 cells to establish TYR-rescued MNT-1 cell lines (Fig. 4A). Whereas re-expression of wt TYR rescued its enzymatic activity and melanin production, re-expression of T373K TYR did not, as expected (Figs. 4B and S6). Notably, rescue with wt TYR decreased tube numbers, but T373K TYR did not affect VM in TYR-KO MNT-1 cells (Fig. 4C and D). These results suggest that the enzymatic activity of TYR is required for regulating VM.