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Role of Presenilin-1 in Aggressive Human Melanoma

Presenilin-1 (PS-1), a component of the gamma (γ)-secretase catalytic complex, has been implicated in Alzheimer’s disease (AD) and in tumorigenesis. Interestingly, AD risk is inversely related to melanoma, suggesting that AD-related factors, such as PS-1, may affect melanomagenesis. PS-1 has been shown to reduce Wnt activity by promoting degradation of beta-catenin (β-catenin), an important Wnt signaling partner. Since Wnt is known to enhance progression of different cancers, including melanoma, we hypothesized that PS-1 could affect Wnt-associated melanoma aggressiveness. Western blot results showed that aggressive melanoma cells expressed significantly lower levels of both PS-1 and phosphorylated-β-catenin (P-β-catenin) than nonaggressive melanoma cells. Immunohistochemistry of human melanoma samples showed significantly reduced staining for PS-1 in advanced stage melanoma compared with early stage melanoma. Furthermore, γ-secretase inhibitor (GSI) treatment of aggressive melanoma cells was followed by significant increases in PS-1 and P-β-catenin levels, suggesting impaired Wnt signaling activity as PS-1 expression increased. Finally, a significant reduction in cell migration was associated with the higher levels of PS-1 and P-β-catenin in the GSI-treated aggressive melanoma cells. We demonstrate for the first time that PS-1 levels can be used to assess melanoma aggressiveness and suggest that by enhancing PS-1 expression, Wnt-dependent melanoma progression may be reduced


Introduction
Melanoma develops from the malignant transformation of melanocytes, the neural crest-derived, pigment-producing cells of the skin [1,2]. Despite making up only 1% of all skin cancers, it accounts for almost the entirety of skin cancer-related deaths, with a dismal 5-year survival rate of only 27% for metastatic disease [1][2][3]. However, with early detection and excision of in situ lesions, the 5-year survival rate of melanoma significantly improves to 99% [1].
The progression of melanoma from in situ, radial growth phase (RGP) to locally infiltrating vertical growth phase (VGP) is multifactorial and influenced by aspects that include but are not limited to: environmental factors such as ultraviolet radiation; genetic mutations of tumor suppressor genes such as TP53; mutations that lead to constitutively active signaling of molecules such as those of the mitogen-activated protein kinase (MEK) pathway; and epigenetic disturbances of gene transcription, such as DNA methylation [4][5][6]. Undoubtedly, this complexity creates a challenge in managing melanoma, as targeting one pathway invariably leads to activation or selection of cells with alternative signaling that continue to drive melanoma progression [5,7,8].
There has been continued interest in exploring how neurodegenerative diseases and associated cellular and microenvironmental factors can affect tumorigenesis. For instance, patients diagnosed with melanoma showed decreased risk for Alzheimer's disease (AD), encouraging research focused on identifying factors involved in pathogenesis of AD that of Wnt signaling [17]. It is reasonable to predict, therefore, that PS-1 may play a role as a potential tumor suppressor by regulating β-catenin availability for nuclear translocation and induction of Wnt signaling in cancer cells.
Here, we investigated the role of PS-1 in melanoma. We showed that increased melanoma aggressiveness was associated with reduced PS-1 expression. Moreover, we demonstrated that increasing PS-1 expression in melanoma cells led to reduced Wnt/βcatenin signaling and migratory potential. These results indicated a novel role for PS-1 as a biomarker for melanoma aggressiveness and repressor of melanoma progression.

PS-1 Expression Was Lower in Aggressive Melanoma Cells with Active Wnt Signaling
We previously showed that nonaggressive melanoma cells exposed to normal skin cells acquired phenotypic and molecular characteristics of increased aggressiveness, which included enhanced cell migration and increased Wnt signaling [33]. Here, WB analysis was performed to ascertain whether PS-1, a known regulator of Wnt function, was implicated in Wnt signaling observed in nonaggressive melanoma cells exposed to normal skin cells. Our WB results ( Figure 1A,B) showed a significant decrease (p < 0.05) in PS-1 expression in the nonaggressive melanoma cell lines WM1552C and UACC1273 (Supplementary Table S1) after exposure to normal human epidermal keratinocytes (keratoCC) and normal human dermal fibroblasts (fibroCC) compared with reference melanoma cells used as control. These results suggest that downregulation of PS-1 expression may be an early event in melanoma cells localized in the skin, and this could facilitate Wnt signaling during the progression of melanoma to more aggressive disease. enhance β-catenin degradation by increasing P-β-catenin levels, which leads to repre of Wnt signaling [17]. It is reasonable to predict, therefore, that PS-1 may play a role potential tumor suppressor by regulating β-catenin availability for nuclear transloc and induction of Wnt signaling in cancer cells.
Here, we investigated the role of PS-1 in melanoma. We showed that increased anoma aggressiveness was associated with reduced PS-1 expression. Moreover demonstrated that increasing PS-1 expression in melanoma cells led to reduced W catenin signaling and migratory potential. These results indicated a novel role for PS a biomarker for melanoma aggressiveness and repressor of melanoma progression.

PS-1 Expression Was Lower in Aggressive Melanoma Cells with Active Wnt Signaling
We previously showed that nonaggressive melanoma cells exposed to normal cells acquired phenotypic and molecular characteristics of increased aggressive which included enhanced cell migration and increased Wnt signaling [33]. Here, WB ysis was performed to ascertain whether PS-1, a known regulator of Wnt function implicated in Wnt signaling observed in nonaggressive melanoma cells exposed to no skin cells. Our WB results ( Figure 1A,B) showed a significant decrease (p < 0.05) in expression in the nonaggressive melanoma cell lines WM1552C and UACC1273 (Su mentary Table S1) after exposure to normal human epidermal keratinocytes (kerat and normal human dermal fibroblasts (fibroCC) compared with reference melanoma used as control. These results suggest that downregulation of PS-1 expression may early event in melanoma cells localized in the skin, and this could facilitate Wnt sign during the progression of melanoma to more aggressive disease. Western blot analysis of PS-1 and P-β-catenin in nonaggressive and aggressive mela cells. Western blot results with corresponding densitometric analysis of WB bands showe creased PS-1 protein expression compared with control cells (reference) in two separate nonag sive melanoma cell lines, WM1552C (A) and UACC1273 (B), after exposure to normal skin ker cytes (keratoCC) and normal dermal fibroblasts (fibroCC) (* p < 0.05). (C) Western blot results corresponding densitometric analysis of WB bands of cell lysates from aggressive melanoma (Sk-Mel28, A375, and C8161) showed lower PS-1 levels than results from nonaggressive mela cells (WM1552C). (D) Western blot results with corresponding densitometric analysis of WB also showed increased Wnt signaling, as demonstrated by the decreased levels of P-β-catenin cat), in the aggressive versus nonaggressive melanoma cells analyzed.
To determine whether PS-1 was in fact decreased in aggressive melanoma, WB ysis for PS-1 expression was performed on lysates from the aggressive melanoma cell Sk-Mel28, A375, and C8161 (Supplementary Table S1). Western blot results showed Sk-Mel28, A375, and C8161 expressed 52%, 29%, and 32%, respectively, of the PS-1 l detected in cell lysates from the nonaggressive melanoma cell line WM1552C (Figure To determine whether PS-1 was in fact decreased in aggressive melanoma, WB analysis for PS-1 expression was performed on lysates from the aggressive melanoma cell lines Sk-Mel28, A375, and C8161 (Supplementary Table S1). Western blot results showed that Sk-Mel28, A375, and C8161 expressed 52%, 29%, and 32%, respectively, of the PS-1 levels detected in cell lysates from the nonaggressive melanoma cell line WM1552C ( Figure 1C). Since lower PS-1 expression could translate into increased Wnt activity, we also analyzed the lysates from the aggressive melanoma cells for P-β-catenin and found that Sk-Mel28, 4 of 12 A375, and C8161 also expressed 54%, 71%, and 57%, respectively, of the P-β-catenin level detected in lysates from WM1552C ( Figure 1D). These results suggest that the lower PS-1 levels detected in the aggressive melanoma cells than in the nonaggressive melanoma cells could explain the relatively higher Wnt signaling activity in the aggressive compared with the nonaggressive melanoma cells.

PS-1 Expression Was Significantly Lower in Advanced-Stage Than in Early-Stage Melanoma
Our in vitro data showing decreased PS-1 expression in melanoma cells as they acquired more aggressive traits suggested that low PS-1 levels could represent a marker for melanoma aggressiveness in vivo. To investigate whether PS-1 expression in melanoma could have clinical significance, IHC staining was performed to assess PS-1 expression in tissue samples from human melanoma at various clinical stages and in normal skin (Supplementary Table S2), used as reference because it is known to ubiquitously express PS-1 [34] (Figure 2A). The mean IHC staining intensity results were grouped into normal skin, early-stage melanoma (Stages I and II), and advanced-stage melanoma (Stages III and IV) ( Figure 2B). These data showed that the mean staining intensity for PS-1 was significantly lower in advanced-stage melanoma (0.77 +/− 0.17, N = 20) than in normal skin (2.1 +/− 0.16, N = 10) (p < 0.01) or early-stage melanoma (1.52 +/− 0.21, N = 35) (p < 0.02). There was no significant difference in mean staining intensity for PS-1 between early-stage melanoma and normal skin. These results suggest that low PS-1 expression could represent a biomarker for increased aggressiveness in melanoma. Since lower PS-1 expression could translate into increased Wnt activity, we also analyzed the lysates from the aggressive melanoma cells for P-β-catenin and found that Sk-Mel28 A375, and C8161 also expressed 54%, 71%, and 57%, respectively, of the P-β-catenin leve detected in lysates from WM1552C ( Figure 1D). These results suggest that the lower PS-1 levels detected in the aggressive melanoma cells than in the nonaggressive melanoma cell could explain the relatively higher Wnt signaling activity in the aggressive compared with the nonaggressive melanoma cells.

PS-1 Expression Was Significantly Lower in Advanced-Stage than in Early-Stage Melanoma
Our in vitro data showing decreased PS-1 expression in melanoma cells as they acquired more aggressive traits suggested that low PS-1 levels could represent a marke for melanoma aggressiveness in vivo. To investigate whether PS-1 expression in mela noma could have clinical significance, IHC staining was performed to assess PS-1 expres sion in tissue samples from human melanoma at various clinical stages and in normal skin (Supplementary Table S2), used as reference because it is known to ubiquitously expres PS-1 [34] (Figure 2A). The mean IHC staining intensity results were grouped into norma skin, early-stage melanoma (Stages I and II), and advanced-stage melanoma (Stages II and IV) ( Figure 2B). These data showed that the mean staining intensity for PS-1 was sig nificantly lower in advanced-stage melanoma (0.77 +/− 0.17, N = 20) than in normal skin (2.1 +/− 0.16, N = 10) (p < 0.01) or early-stage melanoma (1.52 +/− 0.21, N = 35) (p < 0.02) There was no significant difference in mean staining intensity for PS-1 between early-stage melanoma and normal skin. These results suggest that low PS-1 expression could repre sent a biomarker for increased aggressiveness in melanoma.  IHC results showed poor staining for PS-1 in a late-stage melanoma compared with early-stage and normal skin tissue samples. Normal human skin and normal mouse brain tissue sections were used as positive control for PS-1 expression. IHC staining results obtained by omitting the primary antibody (no antibody control) or using an irrelevant isotype immunoglobulin (Ig control) represented negative controls. (B) Box plots show distribution of the IHC staining intensities for the cases of normal skin, early-stage melanoma (Stage I and II), and late-stage melanoma (Stage III and IV). These results showed that staining intensity for PS-1 was significantly lower in late-stage melanoma than in normal skin (* p < 0.01) and early-stage melanoma (** p < 0.02).
antibody (no antibody control) or using an irrelevant isotype immunoglobulin (Ig control) represented negative controls. (B) Box plots show distribution of the IHC staining intensities for the cases of normal skin, early-stage melanoma (Stage I and II), and late-stage melanoma (Stage III and IV). These results showed that staining intensity for PS-1 was significantly lower in late-stage melanoma than in normal skin (* p < 0.01) and early-stage melanoma (** p < 0.02).

Increased PS-1 Expression in DAPT-Treated Aggressive Melanoma Cells Was Associated with Reduced Cell Migration
Wnt signaling has been shown to be involved in the migration and spread of aggressive melanoma [27]. Therefore, cell migration assays were performed to determine if the PS-1-associated decrease in Wnt activity in the DAPT-treated aggressive melanoma cells can lead to a functional effect. Our results showed that DAPT treatment of C8161, A375, and Sk-Mel28, in addition to the increased expressions of PS-1 and P-β-catenin shown above, also led to a significant reduction in cell migration compared with vehicletreated control melanoma cells (C8161, −36% of control; A375, −46% of control; Sk-Mel28, −35% of control) (p < 0.05) ( Figure 4A,B). Moreover, results from MTT cytotoxicity assay showed no significant difference in toxicity between the 15 µM DAPT treatment and control (Supplementary Figure S1). Thus, these findings suggest that the impaired migration of aggressive melanoma cells treated with the 15 µM DAPT was likely the result of a treatment-induced increase in PS-1, with a subsequent increase P-β-catenin levels leading to reduced Wnt signaling.

Increased PS-1 Expression in DAPT-Treated Aggressive Melanoma Cells Was Associated with Reduced Cell Migration
Wnt signaling has been shown to be involved in the migration and spread of aggressive melanoma [27]. Therefore, cell migration assays were performed to determine if the PS-1-associated decrease in Wnt activity in the DAPT-treated aggressive melanoma cells can lead to a functional effect. Our results showed that DAPT treatment of C8161, A375, and Sk-Mel28, in addition to the increased expressions of PS-1 and P-β-catenin shown above, also led to a significant reduction in cell migration compared with vehicle-treated control melanoma cells (C8161, −36% of control; A375, −46% of control; Sk-Mel28, −35% of control) (p < 0.05) ( Figure 4A,B). Moreover, results from MTT cytotoxicity assay showed no significant difference in toxicity between the 15 μM DAPT treatment and control (Supplementary Figure S1). Thus, these findings suggest that the impaired migration of aggressive melanoma cells treated with the 15 μM DAPT was likely the result of a treatmentinduced increase in PS-1, with a subsequent increase P-β-catenin levels leading to reduced Wnt signaling.

Discussion
Presenilins are multipass transmembrane proteins that include the highly homologous PS-1 and PS-2, which play a role in γ-secretase activity and other cell functions [11][12][13]15]. The main difference between PS-1 and PS-2 is that PS-1 is mostly expressed at the cell membrane level while PS-2 is mostly found within the cell and is associated with

Discussion
Presenilins are multipass transmembrane proteins that include the highly homologous PS-1 and PS-2, which play a role in γ-secretase activity and other cell functions [11][12][13]15]. The main difference between PS-1 and PS-2 is that PS-1 is mostly expressed at the cell membrane level while PS-2 is mostly found within the cell and is associated with endosomes and lysosomes. Presenilins were first identified in screening for mutations in patients with familial Alzheimer [12,35]. In fact, it was found that aberrant PS-1 activity was associated with incomplete degradation of amyloid β-peptide, a known contributing factor to the onset of AD [12,13,36]. The interesting finding of lower incidence of certain cancers in patients with AD [9,10], in addition to the PS-1 regulatory function of Wnt signaling, has prompted studies to investigate the role of PS-1 in cancer biology. In some studies, PS-1 was shown to enhance carcinogenesis [37], while other studies, such as in nonmelanoma skin cancer, PS-1 functioned as a tumor suppressor [31]. Thus, the precise role of PS-1 in cancer remains unclear.
The main purpose of this study was to investigate the potential biologic role for PS-1 in melanoma. We recently showed that nonaggressive melanoma cells exposed to normal skin cells increased Wnt signaling and became more aggressive [33]. In this study, we also found that nonaggressive melanoma cells expressed lower PS-1 levels when exposed to normal skin cells compared with control. These results suggest that the increased Wnt signaling and aggressiveness we previously reported for these exposed nonaggressive melanoma cells could be the result of decreased PS-1 expression. In fact, screening cell lysates from several aggressive human melanoma cell lines revealed lower PS-1 expression and increased Wnt signaling, as demonstrated by the reduced P-β-catenin levels compared with nonaggressive melanoma cells. These findings were consistent with previous reports showing the association between loss of PS-1 and increased Wnt signaling and tumorigenesis [31]. To investigate whether PS-1 expression correlated with melanoma aggressiveness in vivo, we performed IHC on human melanoma tissue samples at different clinical stages and found that PS-1 expression was significantly lower in advanced-stage than in early-stage melanoma, demonstrating for the first time that PS-1 could be used as a marker of aggressive melanoma. Moreover, since AD has been associated with aberrant PS-1 function and/or expression [12], our finding that PS-1 levels were lower in patients with certain types of melanoma also provides a possible explanation as to why the incidence of AD is lower in patients with melanoma [9,10].
Given the role of Wnt during tumor progression, it remains an attractive target for anticancer therapy. Thus, preventing Wnt activity in early-stage melanoma could reduce the chances of metastatic spread. For this reason, we explored whether, by increasing PS-1 expression, we could reduce melanoma aggressiveness. DAPT is a GSI previously shown to increase PS-1 expression in treated cells [13]. We reported that aggressive melanoma cells treated for 72 h with 15 µM or 30 µM DAPT had increased PS-1 and P-β-catenin levels. Interestingly, although there was a trend towards lower total β-catenin levels in the treated melanoma cells, this was not significant (data not shown). This suggests that additional time may have been required for our treated aggressive melanoma cells to continue with ubiquitination and proteasomal degradation of the P-β-catenin, which would have resulted in further reduction in total β-catenin. Nevertheless, since only active, nonphosphorylated/nonubiquitinated β-catenin can translocate to the nucleus to induce Wnt-dependent gene transcription [38], the increased P-β-catenin Ser33/37/Thr41 expression observed in our DAPT-treated aggressive melanoma cells could, in itself, be sufficient to negatively affect Wnt-associated functions. In fact, treatment of aggressive melanoma cells with 15 µM DAPT significantly reduced cell migration, a well-known Wnt-regulated activity [16,26], compared with control.
Studies investigating GSI to treat cancers, including melanoma have shown conflicting results. For example, while GSI treatment led to decreased self-renewal and stemness of melanoma cells, long-term treatment facilitated tumor growth, especially in patients with advanced metastatic disease [39]. One explanation as to the inefficacy of certain GSIs in cancer is the negative effects that GSIs have on host antitumor immune activity [40,41]. Another possible explanation of the conflicting data on GSI effects in cancer is the fact that different GSIs have varying pharmacologic and functional profiles depending on the concentrations used [42]. In fact, most clinical trials involve administering relatively high doses of GSI to treat metastatic disease. Some of these doses reach plasma levels that are several orders of magnitude greater than the concentrations used in our study to increase PS-1 expression [43,44]. Thus, treatment of early-stage melanoma with GSIs for enhancing PS-1 expression may require lower doses or shorter treatment regimens, thereby reducing the chances of undesired effects that are associated with longer-term or higher-dose treatment regimes. Our results showed that treatment of aggressive melanoma cells with just 15 µM DAPT could increase PS-1 levels and decrease Wnt signaling and cell migration, suggesting that metastatic potential in melanoma could be reduced by increasing PS-1 expression. Thus, future research aimed at developing treatment strategies to enhance PS-1 expression in aggressive melanoma may reduce melanoma progression.
In summary, our results demonstrated that decreased PS-1 expression was associated with melanoma aggressiveness both in vitro and in human samples. We showed that in aggressive melanoma cells, Wnt signaling activity may increase because of low PS-1 expression. Moreover, we demonstrated that by enhancing PS-1 expression in aggressive melanoma cells, Wnt signaling activity was reduced, and a less migratory and more static phenotype was acquired. Ultimately, these results confirm the importance PS-1in regulating Wnt signaling and suggest a novel role for PS-1 as a biomarker for aggressiveness with therapeutic potential in melanoma.

Cell Cultures
The following cell lines were used (Supplementary Table S1

Serial Coculture
Nonaggressive melanoma cells were exposed in sequence first to normal human epidermal keratinocytes and then to normal dermal fibroblasts as previously described [33]. Briefly, WM1552C or UACC1273 were seeded into transwells (Corning, Corning, NY, USA) containing inserts with porous membranes that allowed for molecular crosstalk between melanoma and normal cells without the cells coming into physical contact. These inserts were then placed in 6-well plates containing the normal epidermal keratinocytes or dermal fibroblasts. The nonaggressive melanoma cells were allowed to grow in sequence first for 24 h with the normal keratinocytes and then for 24 h with the normal dermal fibroblasts. After this coculture sequence, the melanoma cells were harvested for Western blot (WB) protein analysis.

Drug Treatment
In a recent study reporting the effects of g-secretase inhibition in melanoma cells, the authors showed that treatment of cells with DAPT had negligible toxic effects at concentrations ranging from 5 to 60 µM [39]. Based on these results, for this study, it was decided to treat our aggressive melanoma cells with 15 µM and 30 µM DAPT to determine whether these concentrations could have effects on PS-1 expression. Thus, C8161, Sk-Mel28, and UACC1273 were treated were washed once in PBS and then treated with a final concentration of 15 µM or 30 µM DAPT (A8200, Apexbio, Houston, TX, USA) or vehicle control for 72 h. A standard 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) cell viability assay was performed as previously described [45] to determine whether the concentrations of DAPT used resulted in cellular toxicity. Lysates were also obtained from the treated cells to perform WB for the detection of PS-1 and P-β-catenin, as described above.

Immunohistochemistry
Immunohistochemistry (IHC) staining was performed as previously described [45] to assess PS-1 expression in commercially available paraffin-embedded tissue microarray containing normal human skin and melanoma tissue samples at different clinical stages (Me1002b, USBiomax, Rockville, MD, USA). Briefly, after hydration, antigen retrieval, and blocking of endogenous peroxidases and nonspecific binding sites, slides were incubated with primary rabbit anti-PS-1 antibody (1:100, LS-C800364-100, LSBio, Seattle, WA, USA) for 1 h. Then, slides were washed and incubated with secondary antirabbit antibody (PK-4001, Vector Laboratories, Burlingame, CA, USA) for 30 min, washed and incubated with avidin-biotin complex reagent (PK-4001, Vector Laboratories, USA) for 30 min, and washed again and treated with DAB substrate (SK-4105, Vector Laboratories, Burlingame, CA, USA) for stain development. Slides were then washed with distilled water, counterstained with hematoxylin, washed, and dehydrated in a graded ethanol series followed by Histo-Clear. Slides were finally mounted with Aqua-Poly mounting media (Polysciences, Warrington, PA, USA) and observed under light microscopy. To determine appropriate sample size for our IHC study, we assumed 100,000 melanoma patients in the United States [46] and a confidence level of 95% for a difference in PS-1 expression of at least 1/3 (33%). These assumptions indicated that at least 9 samples per group should be analyzed. Since the PS-1 antibody used was specific for mouse and human PS-1, tissue sections from archival paraffin-embedded mouse brain (generous gift from Dr. Maria Traka, Midwestern University, Downers Grove, IL, USA) were used as positive control for IHC PS-1 expression.

Migration Assay
Migration assay was performed as previously described [45]. Briefly, cells were seeded into 3.0 µm pore, 24-well transwell inserts (Falcon, Corning, NY, USA) at 150,000 cells/transwell insert and incubated overnight at 37 • C and 5% CO 2 . Transwell inserts were then cleansed of nonmigrated residue with moist cotton swabs and then further washed in PBS. Transwell inserts were then transferred to wells with crystal violet solution and incubated for 10 min at RT. To extract the stain, stained transwells were then washed with PBS and incubated in wells with 10% acetic acid solution for 10 min. Finally, stain from each sample was transferred to a 96-well plate for OD reading at 590 nm. C8161, A375, and Sk-Mel28 cells were treated with DAPT or vehicle for 72 h prior to the actual migration assay. To acquire images of cell migration, transwell inserts were washed as stated above, and the cells were fixed in methanol for 20 min before staining with crystal violet for 30 s. Next, transwell inserts were washed with PBS. This was followed by cutting of the growth surface (bottom), which was finally mounted on a slide with Cytoseal-60 (Thermo Scientific, Waltham, MA, USA) and observed under light microscopy. A standard 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay was performed as previously described [45] to determine whether the concentration of DAPT used for the migration assay could result in confounding cytotoxicity.

Statistical Analysis
GraphPad statistical software was used to perform t-tests to compare the mean values (+/− standard error of the mean (SEM)) calculated from a minimum of triplicate results from at least two independent experiments between treated and untreated cells or between cocultured and reference control melanoma cells. GraphPad statistical software was also used to perform t-tests to compare the mean intensity IHC staining results (+/−standard deviation of the mean (SD)) among normal skin and early-and advanced-stage melanoma samples. Results showing p values of less than 0.05 (p < 0.05) was considered statistically significant.