CXCR3/ligands are significantly involved in the tumorigenesis of basal cell carcinomas

Abstract

Basal cell carcinoma (BCC) is the most common skin malignancy encountered worldwide. We hypothesized that CXC chemokines, small cytokines involved in inducing directed leukocyte chemotaxis, could play a key role in the modulation of BCC growth. In this study, quantitative RT-PCR revealed that the chemokines CXCL9, 10, 11, and their receptor CXCR3 were significantly upregulated by an average 22.6-fold, 9.2-fold, 26.6-fold, and 4.9-fold, respectively in BCC tissue samples as compared with nonlesional skin epithelium. Immunohistochemistry analysis revealed that CXCR3, CXCL10, and CXCL11, but not CXCL9, colocalized with cytokeratin 17 (K17) in BCC keratinocytes. In addition, CXCR3 and its ligands were expressed in cells of the surrounding BCC stroma. The chemokines and K17 were also expressed in cultured human immortalized HaCaT keratinocytes. Exposure of HaCaT cells or primary BCC-derived cells to CXCL11 peptides in vitro significantly increased cell proliferation. In primary BCC-derived cell cultures, addition of CXCL11 progressively selected for K17+/CXCR3+ co-expressing cells over time. The expression of CXCR3 and its ligands in human BCC keratinocytes, the enhancement of keratinocyte cell proliferation by CXCL11, and the homogeneity of K17+ BCC cells in human BCC-isolated cell population supported by CXCR3/CXCL11 signaling all suggest that CXCR3 and its ligands may be important autocrine and/or paracrine signaling mediators in the tumorigenesis of BCC.

Figure 1

Real-time RT-PCR analysis of the chemokine expression levels in BCCs as compared with nonlesional skin epithelium. BCC tumors (seven nodular BCCs, five superficial BCCs, and five morpheiform BCCs) were studied. The gene expression levels in each BCC subtype were analyzed separately and were calculated in terms of fold change by using the 2^−ΔΔCt equation. The average fold change values of the tissue samples in each of the three BCC subtypes are presented. Error bars represent the range factor difference (2^{−ΔΔCt−ΔCt SD} and 2^{−ΔΔCt+ΔCt SD}). Statistical significance was calculated by Student t test; *P < 0.05.

Figure 2

Serial immunohistochemistry of CXCL9, 10, 11, and CXCR3 in nodular BCCs and nonlesional skin. Five frozen sections of BCC tumors and normal skin tissues were analyzed for the protein expression patterns of CXCL9 (A and B, respectively), CXCL10 (C and D, respectively), CXCL11 (E and F, respectively), and CXCR3 (G and H, respectively). Positive labeling is indicated in red; the frozen sections were counterstained with hematoxylin (blue). Scale bar = 80 μm (A–H).

Figure 3

Dual immunohistochemistry of K17 with CXCL9, 10, 11, and CXCR3 in nodular BCCs. Five frozen BCC biopsies were studied for the colocalization of K17 with CXCL9 (A, E), CXCL10 (B, F), CXCL11 (C, G), and CXCR3 (D, H). K17 is shown as red, whereas CXCR3 and its ligands are indicated as blue. Arrows indicate representative cells dual labeled with K17⁺ co-expression and CXCL10 (F), CXCL11 (G), or CXCR3 (H). Arrowheads indicate representative cells with CXCL9 (E), CXCL11 (G), or CXCR3 (H) labeling in the absence of K17 co-expression in the BCC tumor masses. Scale bar = 80 μm (A–D). Scale bar = 55 μm (E–H).

Figure 4

Quantitative analysis of immunohistochemistry label intensities for CXCL10, 11, and CXCR3 in K17⁺BCC keratinocytes as compared with cells in the stromal area. The mean immuno-labeling intensities of the chemokines were evaluated using Scion Image software. The label intensities were derived from image pixel values (gray scale = 0 to 255) and are presented as mean optical intensity. In each of the 5 BCC biopsies, 10 randomly selected areas from each of the K17⁺ BCC cell nests and the stromal regions were measured and compared for each IHC label by Student t test. *P < 0.05.

Figure 5

Immunohistochemistry analysis of CXCL9, 10, 11, CXCR3, and K17, in HaCaT cells. HaCaT cells were cultured on coverslips until 80% confluency. Cell fixation and permeabilization were conducted, followed by overnight incubation of primary antibodies against CXCL9 (A), CXCL10 (B), CXCL11 (C), CXCR3 (D), and K17 (E). HaCaT cells were counterstained with hematoxylin. The negative control was performed in parallel (F). Scale bar = 45 μm (A–F).

Figure 6

Growth rates of HaCaT cells incubated with CXCL11 for 24 or 48 hours. The y axis represents the mean cell numbers (± SE) of three individual experiments for each peptide concentration as shown on the x axis. Statistical significance of cell number increased relative to untreated controls (0 nmol/L) was calculated by Student t test. *P < 0.05.

Figure 7

Human BCC-derived cell proliferation with or without CXCL11. The y axis represents the cell growth rate, which was calculated by dividing the cell number at the end of the treatment by the cell number seeded initially. A value less than 1 indicates a reduction of the cell population, whereas a value greater than 1 represents the number of times more than the cell number started. The cell growth rates of all of the treatments were compared among each other over 7, 14, and 21 days. Statistical significance was evaluated by analysis of variance. *P < 0.05.

Figure 8

Dual immunohistochemistry of K17 and CXCR3 in human BCC-derived cells incubated with CXCL11. The BCC cells were cultured with 10 nmol/L CXCL11 peptide and subsequently analyzed for the co-expression of K17 and CXCR3 at day 0 (A), 7 (B), 14 (C), and 21 (D–F). K17 labeling is indicated in red, whereas CXCR3 labeling is shown in blue. Scale bar = 50 μm (A–F).

Figure 9

The proportion of cells presenting as K17⁺/CXCR3⁻, K17⁺/CXCR3⁺, K17⁻/CXCR3⁺ in the BCC cell population after incubation with CXCL11. Cells derived from human BCCs were cultured in the presence of 10 nmol/L CXCL11 and examined at day 0, 7, 14, and 21. The y axis represents the percentage of the three cell groups in the cell population at each time point. Statistical analysis was performed by analysis of variance. *P < 0.05.