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. 2024 May 31;25(11):6084.
doi: 10.3390/ijms25116084.

GLI Transcriptional Targets S100A7 and KRT16 Show Upregulated Expression Patterns in Epidermis Overlying the Tumor Mass in Melanoma Samples

Matea Kurtović  1 , Nikolina Piteša  1 , Josipa Čonkaš  1 , Helena Hajpek  1   2 , Majda Vučić  3   4 , Vesna Musani  1 , Petar Ozretić  1 , Maja Sabol  1
Affiliations

GLI Transcriptional Targets S100A7 and KRT16 Show Upregulated Expression Patterns in Epidermis Overlying the Tumor Mass in Melanoma Samples

Matea Kurtović et al. Int J Mol Sci. .

Abstract

Although not completely understood, the role of the Hedgehog-GLI (HH-GLI) signaling pathway in melanoma and epithelial skin tumors has been reported before. In this study, we confirmed in various melanoma cell line models that keratin 16 (KRT16) and S100 Calcium-Binding Protein A7 (S100A7) are transcriptional targets of GLI Family Zinc Finger (GLI) proteins. Besides their important role in protecting and maintaining the epidermal barrier, keratins are somehow tightly connected with the S100 family of proteins. We found that stronger expression of KRT16 indeed corresponds to stronger expression of S100A7 in our clinical melanoma samples. We also report a trend regarding staining of GLI1, which corresponds to stronger staining of GLI3, KRT16, and S100A7 proteins. The most interesting of our findings is that all the proteins are detected specifically in the epidermis overlying the tumor, but rarely in the tumor itself. The examined proteins were also not detected in the healthy epidermis at the edges of the sample, suggesting that the staining is specific to the epidermis overlaying the tumor mass. Of all proteins, only S100A7 demonstrated a statistically significant trend regarding tumor staging and staining intensity. Results from our clinical samples prove that immune infiltration is an important feature of melanoma. Pigmentophages and tumor-infiltrating lymphocytes (TIL) demonstrate a significant association with tumor stage, while mononuclear cells are equally present in all stages. For S100A7, we found an association between the number of TILs and staining intensity. Considering these new findings presented in our study, we suggest a more detailed examination of the possible role of the S100A7 protein as a biomarker in melanoma.

Keywords: HH-GLI; S100A7; biomarkers; epidermis; keratins; melanoma.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
(A) Expression of S100A7 and KRT16 in three melanoma cell lines (CHL-1, A375, and MEL224), after transfection (TF) with GLI1, GLI2, and GLI3 expression plasmids. (B) Validation of S100A7, KRT16, and a known target of GLI proteins, PTCH1, in cell lines CHL-1 and MEL224 resistant to inhibitor GANT61 (GANTR), a model for pathway downregulation. (C) Validation of S100A7 and KRT16 gene expression in the CHL-1 cell line with stable overexpression of SHH, a model of autocrine pathway upregulation. The relative gene expression of S100A7 and KRT16, as well as the PTCH1 gene, is increased, but not statistically significant for S100A7 and KRT16. (D) Validation of S100A7 and KRT16 gene expression in spheroid culture (sph) of the A375 cell line. Dark blue columns show adherent A375 cell line (adh), light purple columns show non-transfected spheroids of A375 cell line, and pink columns show spheroids transfected with GLI1 after 72 h. Data are presented with mean value ± standard deviation (SD), *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.
Figure 2
Figure 2
Association of clinical characteristics with tumor stage in the sample set. Stage is associated with (A) Clark (p < 0.0001), (B) Breslow (p < 0.0001), (C) tumor size (p = 0.003), and (D) number of mitoses/mm2 (p < 0.0001). (E) Association between stage and the presence of preexisting nevus (p = 0.003). (F) Melanoma within a preexisting nevus is detected at a slightly earlier age than melanoma de novo (p = 0.005). (G) Ulceration increases with the tumor stage (p < 0.0001), and (H) regression is most frequently detected in T2 samples but without statistically significant differences between stages (p = 0.183).
Figure 3
Figure 3
Proportion of samples with staining intensities (no expression, weak or strong) for (A) GLI1, (B) GLI3, (C) KRT16, and (D) S100A7 in different melanoma stages (TIS-T4). For each sample, the intensity was measured at three locations: tumor mass (TUMOR), epidermis bordering the tumor mass (BORDER), and epidermis overlying the tumor mass (CENTRAL). In general, the staining intensities for all proteins were stronger in the epidermis (both border and central) compared to the tumor mass.
Figure 4
Figure 4
Representative staining of the border area of the T4 tumor for (A) GLI1, (B) GLI3, (C) KRT16, and (D) S100A7. (E) Negative control (no primary antibody), and (F) higher magnification of GLI1 staining in the tumor. The scale bar for (A–E) was 200 μm and for (F) was 50 μm.
Figure 5
Figure 5
Different localizations of GLI staining. GLI3 shows stronger staining in the basal layer in the epidermis of low-stage melanoma, while GLI1 intensity is uniform through the epidermis overlying the tumor. NC denotes negative control. The scale bar for the upper panel was 100 μm, and for the lower panel, it was 50 μm.
Figure 6
Figure 6
Comparison of protein expressions (A) GLI1 vs. GLI3, (B) GLI1 vs. KRT16, (C) GLI1 vs. S100A7, and (D) S100A7 vs. KRT16 in the same location (epidermis bordering the tumor = BORDER). There is a significant association between GLI1 expression and expressions of all other tested proteins for border vs. border (p < 0.0001). (E–H) Expression of the same proteins in BORDER vs. tumor mass (TUMOR). GLI1 (E), GLI3 (F), and S100A7 expression (G) in the tumor tissue is significantly associated with the expression in the border epidermis (p < 0.0001 for all three proteins), while KRT16 expression (H) is not significantly associated (p = 0.107).
Figure 7
Figure 7
Distribution of intensities of staining for GLI1 (A), GLI3 (B), KRT16 (C), and S100A (D) in the epidermis bordering the tumor (BORDER) by stage (TIS, T1, T2, T3, and T4). All the stained proteins show significant differences between different stages (p < 0.0001 for all).
Figure 8
Figure 8
The role of immune infiltration in melanoma. Immune cells were determined as follows: none = 0, few = 1, medium/focally dense = 2, and dense = 3. Mononuclear cells are equally present in all stages (TIS, T1, T2, T3, and T4) (A) (p = 0.326), while pigmentophages (B) (p = 0.015) and TIL (C) (p < 0.0001) demonstrate a significant association with tumor stage. Mononuclear cells and pigmentophages are significantly associated with regression (p = 0.028 and p = 0.05, respectively) (D,E), and a similar trend is visible for TIL (F), but their value is not statistically significant (p = 0.238). (G) Association of S100A7 staining in the border epidermal layer and number of TIL (p = 0.013).
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