3, 3′- (3, 5-DCPBC) Down-Regulates Multiple Phosphokinase Dependent Signal Transduction Pathways in Malignant Melanoma Cells through Specific Diminution of EGFRY1086 Phosphorylation

Melanoma is the most dangerous skin malignancy due to its strong metastatic potential with high mortality. Activation of crucial signaling pathways enforcing melanoma progression depends on phosphorylation of distinct tyrosine kinases and oxidative stress. We here investigated the effect of a bis-coumarin derivative [3, 3′- ((3″, 5′-Dichlorophenyl) methylene) bis (4-hydroxy-2H-chromen-2-one)] [3, 3′- (3, 5-DCPBC)] on human melanoma cell survival, growth, proliferation, migration, intracellular redox state, and deciphered associated signaling pathways. This derivative is toxic for melanoma cells and non-toxic for melanocytes, their benign counterpart, and fibroblasts. 3, 3′- (3, 5-DCPBC) inhibits cell survival, migration, and proliferation of different metastatic and non-metastatic melanoma cell lines through profound suppression of the phosphorylation of Epidermal Growth Factor receptor (EGFR) and proto-oncogene cellular sarcoma (c-SRC) related downstream pathways. Thus, 3, 3′- (3, 5-DCPBC) endowed with the unique property to simultaneously suppress phosphorylation of multiple downstream kinases, such as EGFR/JAK/STAT and EGFR/SRC and their corresponding transcription factors.


Introduction
Malignant melanoma represents the most aggressive and deadliest form of skin cancer [1][2][3]. Several systemic therapies (cytotoxic chemotherapy, targeted drugs, immunotherapy, hormonal therapy, radiation therapy, and bio-chemotherapy) have been approved by the US Food and Drug Administration (FDA) [4,5]. Surgery still represents the first treatment option for primary melanoma where metastasis has not yet occurred. At later metastatic stages, systemic therapies are mandatory. The most successful treatment options against non-resettable metastatic melanoma are immunotherapies with antibodies directed against CTLA-4 and PD-1 [6,7] or their combination (small kinase and checkpoint inhibitors) [8][9][10]. The overall response rate of 10% to 15% for anti-CTLA-4 (ipilimumab), 25% to 45% for anti-PD-1 (nivolumab or pembrolizumab), and around 60% for their combination were embraced as a recent breakthrough in the therapy of a previously hard-to-treat malignancy [11]. However, due to the resistant nature of malignant melanoma [12], 40% of the patients do not respond to the combined therapy [13]. Due to severe and in part lifethreatening side effects of immune checkpoint inhibitors affecting up to 50% of melanoma patients treated with anti-CTLA-4 immunotherapy [14], and even more when subjected to a combined anti-CTLA-4 and anti-PD-1 therapy [15], there is an urgent quest for new strategies in the battle against metastatic melanoma. Several promising agents have been tested against single protein kinases in malignancies [16,17].
Phosphorylated-receptors of tyrosine/serine/threonine protein kinases and their downstream targets are prime molecular players enforcing cell growth, survival, proliferation, and migration [18,19]. A number of these effectors within distinct signaling pathways are hyperphosphorylated and, consequently, hyperactivated in different cancers, including melanoma [20,21]. Some of the new therapeutic interventions against these kinases have successfully reached clinical trials, while others have already been approved by the US-FDA and entered clinical routine [22][23][24][25]. Currently, no single systemic therapy has successfully prevented melanoma progression in the long term. Due to the emergence of resistance against these drugs, multiple phase II and III melanoma trials are currently underway that are either studying the effect of combination treatments or striving for synthetic and natural compounds [26,27]. Combined therapies targeting two tyrosine kinases at least delay the development of resistance [28]. A newly emerging concept for treating advanced malignant melanoma is based on uncovering synthetic compounds targeting multiple signaling pathways and their corresponding genes.
Bis-coumarins, a benzophenone family of natural compounds, have been reported to exhibit antioxidant, anti-inflammatory, anti-microbial, anti-glycation, anti-leukemic, and urease inhibition activities. They also have previously been studied as potential drug candidates against normal, non-cancerous, as well as various types of cancerous cancers cell lines [29]. They are, however, not endowed with the required potential to simultaneously target several signaling pathways in melanoma cells. We here set out to evaluate the effect of a bis-coumarins derivative, [3,3 -((3,5-dichlorophenyl)methylene) bis (4-hydroxy-2Hchromen-2-one)] [3, 3 -(3, 5-DCPBC)] on growth, survival, proliferation, and migration of non-metastatic and metastatic melanoma cells and to investigate its underlying molecular mode of action. In the present study, we demonstrated that treatment of human metastatic and non-metastatic melanoma cells with 3, 3 -(3, 5-DCPBC) profoundly diminished phosphorylation of the Epidermal Growth Factor Receptor (EGFR), proto-oncogene cellular sarcoma (c-SRC) and simultaneously suppressed phosphorylation of key downstream effectors and signaling pathways known to enforce melanoma progression. We also uncovered the non-toxic nature of this compound against human melanocytes, the benign counterpart of malignant melanoma cells. In aggregate, we here report for the first time a novel anti-melanoma drug candidate that not only suppresses cellular functions, key for melanoma progression but simultaneously targets multiple phosphor-tyrosine kinases. This is a clinically relevant advancement, and thus 3, 3 -(3, 5-DCPBC) may hold the unique therapeutic potential to be further tested in preclinical and clinical studies.

3, 3 -(3, 5-DCPBC) Diminishes Metastatic Melanoma Cell Proliferation
The effect of 3, 3 -(3, 5-DCPBC) was further explored on the proliferation of metastatic A375 melanoma cells as a prime event in melanoma progression. For this purpose, BrdU incorporation was studied as previously described [34]. The DNA thymidine analog 5-Bromo-2 -deoxyuridine (BrdU) was incorporated into rapidly growing metastatic melanoma cells during the S phase of the cell cycle in the presence and absence of 3, 3 -(3, 5-DCPBC) and was detected fluorometrically by an antibody directed against BrdU at 548 and 576 nm excitation and emission, respectively. 3, 3 -(3, 5-DCPBC) efficiently suppressed melanoma cell proliferation at 48 h of treatment ( Figure 3a). This anti-proliferative 3, 3 -(3, 5-DCPBC) effect corresponds to an average of the relative fluorescence of 1, compared to 4.9 of the DMSO control at the same time point (Figure 3a).
In a complementary approach, BrdU positive cells from immunofluorescence photomicrographs were quantitated, and the percentage of BrdU positive cells were significantly reduced ( Figure 3b). These data indicate a strong anti-proliferative effect of compound 3, 3 -(3, 5-DCPBC) on A375 metastatic melanoma cells.

Molecular Mechanism of Action of 3, 3 -(3, 5-DCPBC)
A phospho-proteome profiling array [39] was employed to gain insight into the mechanism for 3, 3 -(3, 5-DCPBC) inhibiting melanoma cell survival, growth, proliferation, and migration; 1 × 10 5 A375 melanoma cells were treated with 3, 3 -   A list of non-affected kinases are shown in Supplementary Tables S1 and S2. Densitometric analysis of significant phosphorylation sites of 25 kinases and their associated p values is shown in Figure 4e. Sixteen different kinases were more than 2-fold downregulated upon treatment with 3, 3 -(3, 5-DCPBC) compared to DMSO as represented by the blue color (Figure 4f). The MA plot depicts Log2 fold changes on the y-axis, and Log2 mean expression on the x-axis (Figure 4f). Eleven kinases did not show any significant changes. Among the down-regulated phosphokinases, predominantly members of the family of the tyrosine kinase family were suppressed, phosphorylation of serine or threonine kinases was less frequently affected ( Figure 4g). The y-axis represents the fold change, and the x-axis represents the position of the phospho-sites in the gene amino acid sequence (Figure 4g). Of all the kinases significantly downregulated by 3, 3 -(3, 5-DCPBC), the tyrosine kinase phospho-motif (blue filled circles) is the most affected phospho-motif in A375 melanoma cells treated with 3, 3 -(3, 5-DCPBC) as compared to threonine (green filled circles) or serine (pink filled circles). Phospho-motif mapping thus allowed us to uncover major kinases and their key phosphorylation sites that likely are involved in uncontrolled signaling in melanoma progression. Pathway analysis was further employed for the most suppressed kinases with downregulation of phospho-motifs to explore which signaling pathways are most prominently affected (Figure 5a). The x-axis represents the gene ratio, which is presented as % of total differential gene expression (DGE) in all GO clusters, and the y-axis represents the associated pathway. The color refers to the p-value and the count as the number of occurrences of this gene per GO cluster. Pathway analysis with the differentially down-regulated phosphor-motifs depicts receptor tyrosine kinases, inflammatory interleukins, ERK1/2, AKT, and mTOR as the major signaling cascades that are affected after treatment of melanoma cells with 3, 3 -(3, 5-DCPBC) for a period of 4 h. Briefly, phosphor tyrosine modulation at a specific residue of Epidermal Growth Factor Receptor (EGFR Y1086 ) and Fc-gamma Receptors Y412 , and their downstream targets JAK/STAT were the most suppressed targets, including STAT2 Y689 , STAT5α Y697 , STAT5α/β Y694/Y699 , and STAT6 Y641 . Among the other 3, 3 -(3, 5-DCPBC) suppressed pathways, mTORS 2448 , ERK1/2 Y204/Y187 , SRC Y419 , and β-Catenin Y654 were identified as highly important for melanoma progression. To understand whether inhibition of these phosphorylation sites following treatment of A375 melanoma cells with 3, 3 -(3, 5-DCPBC) will persist, phosphokinase array analysis was performed from lysates of A375 melanoma cells, which had been treated with 3, 3 -(3, 5-DCPBC) for 18 h (Figure 5b). The normalization of phosphoproteins expression by the β actin expression for 4 and 18 h depicts that downregulation of phosphorylation sites as observed at 4 h persists in many previously identified phosphokinases at 18 h.  Figure 4. Densitometry data were obtained using gel quant software and normalized for 4 and 18 h with β-actin. Results are expressed as "Arbitrary Density units" and presented a mean ± S.D. for 3 independent experiments. Data were analyzed by performing Two-way ANOVA, and the statistical difference is shown as * p values. The table shows a list of selective kinases whose phosphory- A total of 12 phosphorylation sites of nine different phosphoprotein kinases were found to be persistently downregulated both at 4 and 18 h (Figure 5b,c). These data imply that they constitute likely targets for 3, 3 -(3, 5-DCPBC) Treatment of A375 melanoma cells with 3, 5 DCPBC for 4 and 18 h profoundly suppressed phosphorylation of critical targets like EGFR, SRC, STAT3, JNK1/2, and MSK. Mainly phosphorylation sites of tyrosine kinases were suppressed (Figures 4g and 5c) and, consequently, attenuated those target effectors downstream of tyrosine kinase signaling, which enforces melanoma progression.

Discussion
The significant unprecedented finding of this study is that the bis-coumarin derivative 3, 3 -(3, 5-DCPBC) has profound inhibitory properties on critical steps of malignant melanoma progression. Accordingly, proliferation, migration, and survival of melanoma cells-by contrast to benign melanocytes and fibroblasts-are impressively downregulated in-vitro in the presence of moderate concentrations of 3, 3 -(3, 5-DCPBC).
Although, enhanced ROS concentrations have been reported to be associated with melanoma progression [35][36][37]. However, we found that the strong effect of 3, 3 -(3, 5-DCPBC) on critical features of melanoma progression is not due to antioxidant properties but is instead a consequence of its outstanding suppressive potential of different important tyrosine phosphokinases involved in melanoma progression ( Figure 6). Phosphokinases modulate several cellular functions [40], and activation of multiple phosphokinases enforces the progression of melanoma and many other malignancies [41]. In particular, autophosphorylation of EGFR with phosphorylation of the downstream pathways play prime roles in melanoma progression [42]. Of note, 3, 3 -  Table S1). This is most interesting as phosphorylation of this EGFR residue regulates various signal transduction pathways (mTOR, SFK, JAK-STAT, MSK, ERK) involved in cell proliferation, cell migration, and cell survival. Notably, these downstream pathways are hyperphosphorylated in various cancers, including melanomas [43][44][45][46][47]. In addition, hyperphosphorylation of mTOR at its threonine Thr2446 and serine residues (Ser2448 and Ser2481) occurred both via the EGFR-ERK-S6K1 axis and the PI3K/AKT axis [48] and is related to growth in various types of cancers and melanoma [49]. We further explored downstream pathways and the associated genes that are regulated through EGFR Y1086 phosphorylation (Figure 5a). Of note, EGFR/JAK/STAT and EGFR/SRC tyrosine kinases, the most important among them EGFR and SRC, and their downstream effectors were markedly downregulated upon 3, 3 -(3, 5-DCPBC) treatment as confirmed by downregulation of 16 genes coding for distinct phosphotyrosine kinases (Figure 4f). Suppression of phosphorylation (activation) of the mTOR pathway (mTOR, PRAS40, and ERK1/2/3) was observed mainly after 4 h of 3, 3 -(3, 5-DCPBC) treatment. EGFR Y1086 phosphorylation can activate the SRC family of kinases (SFKs) [50]. Tyrosine phosphorylation of members of SKFs plays a key role in cell differentiation, motility, proliferation, and survival [50][51][52]. Remarkably, we found profound suppression of phosphorylation of SKF members, among them SRC Y419 , LYN Y397 , LCK Y394 , FYN Y420 , YES Y426 , FGR Y412 , HCK Y41 , and FAK Y319 in A375 malignant melanoma cells after treatment with 3, 3 -(3, 5-DCPBC). Most excitingly, it is endowed with the potential to target multiple kinases to prevent cellular migration (via SRC), proliferation, and protein synthesis (via AKT/mTOR). The Red circular outline indicates the post-translationally regulated phosphokinase sites which were found to be significantly down-regulated in melanoma cells after 3, 3'-(3, 5-DCPBC) treatment compared to controls. The ash-colored circular nodes represent intermediate signaling pathway transcription factors whose phosphorylation site was not changed. They are mentioned to depict the complete pathways. The yellow, green, and pink phosphorylation sites represent tyrosine, threonine, and serine, respectively. This is most interesting as phosphorylation of this EGFR residue regulates various signal transduction pathways (mTOR, SFK, JAK-STAT, MSK, ERK) involved in cell proliferation, cell migration, and cell survival. Notably, these downstream pathways are hyperphosphorylated in various cancers, including melanomas [43][44][45][46][47]. In addition, hyperphosphorylation of mTOR at its threonine Thr2446 and serine residues (Ser2448 and Ser2481) occurred both via the EGFR-ERK-S6K1 axis and the PI3K/AKT axis [48] and is related to growth in various types of cancers and melanoma [49]. We further explored downstream pathways and the associated genes that are regulated through EGFR Y1086 phosphorylation (Figure 5a). Of note, EGFR/JAK/STAT and EGFR/SRC tyrosine kinases, Figure 6. 3, 3 -(3, 5-DCPBC) impacts important regulatory hubs in melanoma progression. Summary scheme depicting 3, 3 -(3, 5-DCPBC) targeting EGFR and SRC kinases, AKT/mTOR, and RAS/RAF/MAP kinases. Taken together, 3, 3 -(3, 5-DCPBC) shows excellent tyrosine kinase and serine kinase inhibiting properties. Most excitingly, it is endowed with the potential to target multiple kinases to prevent cellular migration (via SRC), proliferation, and protein synthesis (via AKT/mTOR). The Red circular outline indicates the post-translationally regulated phosphokinase sites which were found to be significantly down-regulated in melanoma cells after 3, 3 -(3, 5-DCPBC) treatment compared to controls. The ash-colored circular nodes represent intermediate signaling pathway transcription factors whose phosphorylation site was not changed. They are mentioned to depict the complete pathways. The yellow, green, and pink phosphorylation sites represent tyrosine, threonine, and serine, respectively.
Our data on the importance of 3, 3 -(3, 5-DCPBC) induced suppression of EGFR Y1086 phosphorylation is further underscored by the recent finding that growth hormone via EGFR and SRC kinases upregulates the transcription factor c-Kit for melanogenesis, and more importantly, that mutated Kit enforces MITF (Microphthalmia-associated tran-scription factor)-dependent transcription programs in melanoma [53,54]. Finally, recent data on the prime role of the tyrosine kinases SRC and the AKT/mTOR axis in melanoma progression [55,56] highlights the unique implication of 3, 3 -(3, 5-DCPBC) as a promising small molecule drug candidate. The finding that 3, 3 -(3, 5-DCPBC) is endowed with the unique property to simultaneously suppress many phospho-tyrosine kinases further underscores its potential prime clinical relevance. Interestingly, simultaneous inhibition of tyrosine phosphorylation of EGFR and SRC kinases can overcome the frequently developing BRAF resistance in melanoma [57,58]. Finally, phosphorylation on Y705/727 and Y694/699 of STAT3 and STAT5α/β most likely in cooperation with MSK enforce phosphorylation of ERK1/2 (extracellular signal-regulated kinase) or p38 in a growth factor receptor-independent signaling pathways [59]. These pathways are essential for melanoma progression, and their inhibition by 3, 3 -(3, 5-DCPBC) likely suppresses melanoma progression.

Cell Culture
Metastatic melanoma cell lines; A375, SK-Mel-28, non-metastatic melanoma cell line; WM-266-4, WM115, and FF95 fibroblasts were grown in DMEM supplemented with Pen/Strep and 20% FBS at 37 • C, and 5% CO 2 . Cells were synchronized with starving medium (0.2% F-10 Nut Mix Ham 1X) for a further 12 h. After that, cells were either incubated with 3, 3 -(3, 5-DCPBC), or w/o in a control group, incubated with identical volumes of DMSO in DMEM for the indicated concentrations and incubation times. Human primary melanocytes were grown in M2 media.

Cell Cytotoxicity Assay
The MTT assay was employed to evaluate cytotoxicity as previously described [60]. Melanoma cells and their benign control cells were grown in 96 well plates, as described in Section 2.2. MTT solution was added for 4 h, and formazan crystals were dissolved in DMSO for 15 min at room temperature. Absorbance was recorded at 550 nm, and the reference wavelength was set to 650 nm using a microplate reader (Varioskan Lux, Thermofisher Scientific). Cytotoxicity of 3, 3 -(3, 5-DCPBC) was calculated as the relative ratio of optical densities compared to non-treated controls.

Transwell Migration Assay
The Transwell ® migration assay was performed as earlier described, with few modifications [61][62][63]. In brief, 1 × 10 5 cells in 200 µL of either starved medium or 20% FBS in DMEM were loaded onto 8-micrometer pore Transwell inserts (upper chambers) and incubated for 2 h at 37 • C in 5% CO 2 . The lower chambers were loaded either with 600 µL of 20% FBS in DMEM, chemotactic stimuli (human placental type IV collagen) at a concentration of 100 µg/µL or dissolved in 20% FBS in DMEM, and incubated for 2 h at 37 • C in 5% CO 2 . Thereafter, cells were fixed and stained using the Diff Quik ® Stain kit. Non-migrated cells were removed from the upper chambers using cotton swabs. Perforated filters were removed and fixed on microscopic slides. An average number of migrated cells was counted in five high-power microscopic fields (HPF), randomly chosen at 200X magnification from each of the three technical and four biological replicates. Images of cells that migrated to the downside of the perforated membrane were collected using a Nikon TE300 inverted epifluorescence microscope.

Bromodeoxyuridine (BrdU) Incorporation Assay
Melanoma and control cells were seeded in 96-well plates as described in Section 2.2, followed by the treatment of 1 µM 3, 3 -(3, 5-DCPBC)/100 µL of 20% FBS in DMEM in each well for different incubation times. After incubation, 3 µg/µL BrdU was added to each well for another 2 h at 37 • C, followed by fixation of cells with 1:1 acetone solution for 30 min. DNA strains were denatured with 2N HCl, and non-specific binding sites were blocked by 5% BSA dissolved in TBST. 50 µL primary anti-BrdU antibody was suspended in TBST at a dilution of 1:10 and added to detect the incorporated BrdU dye. Melanoma cells and control cells in each well were then treated for 2 h with 50 µL Alexa Fluor conjugated 555 goat-anti-mouse secondary antibodies diluted in TBST at 1:200 dilution. After washing melanoma and control cells with TBST buffer, fluorescence in each well containing 100 µL TBST was measured at 548 nm excitation and 576 nm emission.

BrdU Immunofluorescence Staining
Cells were grown in Millicell EZ chamber slides coated with poly-l-lysine and were further incubated with 100 µM BrdU for 2 h. Melanoma cells and control cells were fixed with 1:1 acetone solution for 30 min, DNA was hydrolyzed with 2N HCl for 30 min, and non-specific binding sites were blocked by 5% BSA dissolved in TBST. Melanoma cells and control cells were incubated overnight with 500 µL primary anti-BrdU antibody in TBST at a 1:10 dilution, washed thrice with TBST buffer. After that, cells were treated with 500 µL, Alexa Fluor conjugated 555 goat anti-mouse secondary antibody suspended in TBST at a 1:200 dilution in each well for 2 h at room temperature. Cell nuclei were stained with DAPI (1 µL/mL of DAPI in PBS) for 10 min, cells were fixed with formaldehyde and mounted with Fluoromount (Dako). Melanoma and control cells were examined under a fluorescence microscope at 40× magnification with AxioVision A5 microscope (Zeiss Inc., Oberkochen, Germany). BrdU-positive melanoma and control cells were counted manually from three independent images. The percentage of proliferating cells was assessed by calculating the number of BrdU-positive cells in the 3, 3 -(3, 5-DCPBC) treated group, and the DMSO treated control group in 5 to 6 random fields.

Messurement of Intracellular Radical Scavenging Activity
Intracellular cytosolic and mitochondrial superoxide radical scavenging activity was measured through fluorimeter using MitoSOXTM Red and DHE dyes. Briefly, 105 A375 cells grown in DMEM media were further starved in F-10 Nut Mix (Ham) for a period of 12 h, thereafter, media was aspirated, and 100 µL of sample solution (1 µM of each test compound dissolved DMEM) was added in each well of the 96-well plate. After 12 h. incubation cells were washed thrice with PBS and treated with 2.5 µM MitoSOXTM Red and 5 µM DHE dyes for 15 and 20 min at 37 • respectively. After incubation, the cells were rinsed with PBS and to each well was added 100 µL buffer (PBS with freshly added 5 mm glucose). Finally, DHE fluorescence was measured at 510 nm excitation, and 595 nm emission, and MitoSOXTM Red fluorescence was measured at 510 nm excitation, and 595 nm emission. In both assays rotenone and N-acetylcysteine and rotenone were used as positive and negative controls respectively. However, DMSO was used as vehicle control.

The Human Phospho-Kinase Array Assay and Western Blot Analysis
The phosphorylation level of kinases was determined with the Proteome Profiler Array Kit (R&D Systems) according to the manufacturer's instructions. Protein concentrations were determined by the Bradford protein assay. To block non-specific sites, each membrane was incubated in an array blocking buffer for 1 h. 3, 3 -(3, 5-DCPBC), and DMSO treated cell lysates (334 µL cell lysate/1 mL of array buffer corresponding to 200 µg protein lysate) were applied on membranes and incubated overnight. Thereafter, membranes were washed with 1X washing buffer followed by incubation with 20 mL of the detection antibody for 2 h on a shaker at room temperature. Membranes were thoroughly rinsed with washing buffer thrice and further incubated with Streptavidin-HRP for 30 min at room temperature. Membranes were washed with 1× washing buffer for 10 min, and after that, all membranes were simultaneously exposed to SignalFire plus chemiluminescent reagents for 1 min. Phospho-kinase array data were developed on Vilber FusionFx Chemiluminescence Imager for 1 to 10 min with multiple exposure times.

Statistical Analysis
Graph Pad Prism software 5 (GraphPad Software, Inc., San Diego, CA, USA) was used to analyze data. Parametric one-way analysis of variance was used with Tukey's posthoc analysis to compare multiple groups of 3, 3 -(3, 5-DCPBC) treated, and DMSO treated groups. Two-way analysis of variance was used with Tukey's posthoc test to compare two different time points and groups of 3, 3 -(3, 5-DCPBC) treated, and DMSO treated groups. The Student's t-test (two-tailed) was employed to compare the two groups of counted cells taken from representative photomicrographs. Significance was defined as p < 0.05. p values were assigned * with p < 0.01, ** with p < 0.001 and *** with p < 0.0001. R studio version 1.1.463 was used for data analysis and networking analysis, respectively.

Conclusions
In conclusion, with the urgent need for new therapeutics to target multiple tyrosine kinases as an emerging concept for advanced melanoma, 3, 3 -(3, 5-DCPBC) might hold promise for an efficient alternative approach to currently established therapies [49,57].
We discovered that 3, 3 -(3, 5-DCPBC) is highly suppressive on many steps and pathways of melanoma cell progression at very low concentrations. At the same time, it is non-toxic for non-tumorous melanocytes and fibroblasts. The efficient combined targeting of EGFR, SRC, STAT, and MAPK by 3, 3 -(3, 5-DCPBC) in melanoma cells may long term assist clinicians to prevent melanoma growth and even to overcome drug resistance. Further preclinical data are now required to use this compound in future clinical trials.