Performed the experiments: BM. Analyzed the data: BM RLG. Contributed reagents/materials/analysis tools: BM SJ MG JG TH. Wrote the paper: BM RLG.
The authors have declared that no competing interests exist.
Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) are the most frequent skin cancers in humans. An intact immune system is critical for protection against SCC since organ transplant recipients (OTR) have a 60- to 100-fold higher risk for developing these tumors. The role of the innate immune system in tumor immunosurveillance is unclear. Our aim was to determine the expression of selected innate immune genes in BCC and SCC arising in immunocompetent and OTR patients. Lesional and peri-lesional skin from 28 SCC and 19 BCC were evaluated for mRNA expression of toll-like receptors (TLR) 1–9, downstream TLR signaling molecules, and antimicrobial peptides. 11 SCC occurring in OTR patients were included in the analysis. We found that SCC but not BCC showed significantly elevated expression of TLRs 1–3, 5–8, TRIF and TRAF1. TNF was increased in SCC compared to normal skin. BCC showed increased IFNγ. hBD1, hBD2 and psoriasin mRNA and protein expression were significantly higher in SCC than in normal skin and higher than in BCC. SCC from OTR showed only an increase in hBD2 but no increase in hBD1 or psoriasin. We conclude that innate immune gene expression in SCC is distinct from normal skin and BCC. BCC shows lesser induction of innate immune genes. SCC from OTR patients have depressed expression of hBD1 and psoriasin compared to SCC from immunocompetent patients.
Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are the most frequent non-melanoma skin cancers (NMSC) in humans
Currently, most research on tumor immunosurveillance in NMSC, including those within the framework of cancer immunoediting
The activation of an innate immune response has diverse consequences. AMPs can kill microbes and prevent infection
Since very little data has been published on innate immune responses in NMSC we wished to provide an overview of innate immune gene expression in a population of patients with SCCs and BCCs, focusing on TLRs, TLR-signaling intermediates, cytokines and antimicrobial peptides. Since OTRs have a significantly higher risk for SCC
A total population of 37 patients was included in this study, comprising 28 patients with SCC and 19 with BCC. SCCs came from 17 immunocompetent patients and 11 that were OTR. Patient demographics in each group were similar with the exception of OTR who were generally younger (
1A) Patients enrolled in the study, tumor diagnoses and immune status. SCC = squamous cell carcinoma. BCC = basal cell carcinoma. Imm’comp. (ICP) = immunocompetent patients. Imm.sup.(OTR) = organ transplant recipients (immunosuppressed patients). Gender (percentage male) and age (mean ± SEM) distribution in each group of patients is shown. 1 B) Age distribution in patient groups. Scatter plots including mean ± SEM are shown. *p < 0.05, ***p < 0.005 (one-way ANOVA with Bonferroni‘s post-test). 1 C) From each case, a specimen was collected from the center of the tumor (T), from the tumor margin (M) including tumor cells as well as non-tumor cells, from peritumoral tissue (P) that does not contain any tumor cells, and from normal control skin (N) in far distance from the tumor.
Cutaneous SCC typically retains characteristics of squamous differentiation. To further characterize the specimens in our study, we measured mRNA expression of several epidermal differentiation genes. Samples within the central tumor tissue of SCC showed significantly elevated mRNA expression of filaggrin (13.60±4.52-fold (mean ± SEM), p<0.05) and keratin 10 (10.83±0.51-fold, p<0.001) compared to normal skin. Filaggrin and keratin 10 expression in the tumor center was also higher compared to the tumor margin (0.44±0.22, p<0.001; 1.56±0.51, p<0.05) and to peritumoral tissue (0.62±0.18, p<0.001; 1.19±0.34, p<0.01). Filaggrin and keratin 10 expression in the tumor center of SCC was also significantly higher than in the tumor center of BCC (5.50±2.98, p<0.05; 0.39±0.09, p<0.001). For loricrin, reduced mRNA expression in the tumor margin of SCC was observed compared to normal skin (0.073±0.021, p<0.001), the tumor center (8.29±5.29, p<0.001) and peritumoral tissue (0.33±0.08, p<0.05) (
mRNA expression of differentiation gene loricrin (LOR), filaggrin (FLG), keratin 5 (K5) and keratin 10 (K10) in the tumor center (T), tumor margin (M), peritumoral tissue (P) and distant normal skin (N) in cutaneous squamous cell carcinoma (SCC, black bars) and basal cell carcinoma (BCC, white bars). Relative mRNA expression to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and normalized to normal skin is shown. Depicted are mean ± SEM values. *p<0.05, **p<0.01, ***p<0.001 by Kruskal-Wallis test with Dunn’s post-test for comparison between N, P, M and T and by Mann Whitney U test for comparison between SCC and BCC in each group. N = 28 SCC and 19 BCC.
TLR1 mRNA expression in the SCC tumor center was 5.49±1.21-fold higher (p<0.01) than in normal skin and also significantly higher than in the tumor center of BCCs (1.82±1.21, p<0.001). TLR2 mRNA expression in the SCC tumor center was 7.90±1.76-fold higher (p<0.01) than in normal skin and also higher than in the tumor center of BCCs (1.41±0.35, p<0.001). TLR3 mRNA expression in the SCC tumor center was 8.51±2.11-fold higher (p<0.01) than in normal skin and also higher than in peritumoral tissue (1.61±0.70, p<0.05). TLR5 mRNA expression in the SCC tumor center showed the largest magnitude difference and was 30.8±22.9-fold higher (p<0.05) than in normal skin and also significantly higher than in the tumor center of BCCs (0.92±0.46, p<0.01). TLR6 mRNA expression was significantly higher in the SCC tumor center (4.48±1.44) than in the tumor margin (0.96±0.48, p<0.05). TLR7 mRNA expression in the SCC tumor center was 15.6±3.70-fold higher than in normal skin (p<0.001) and also significantly higher than in peritumoral tissue (3.37±1.35, p<0.01) and than in the tumor center of BCCs (1.98±0.53, p<0.001).
Of note, TLR8 mRNA expression was significantly higher in the peritumoral tissue of SCC (13.93±0.57) as well as in the tumor center (7.46±4.99) compared to normal skin (p<0.05 for both) and significantly higher compared to BCC peritumoral (0.36±0.21) and BCC tumor center tissue (0.35±0.14) (p<0.01 for both) (
mRNA expression of toll like receptor genes 1–9 (TLR1-9) in the tumor center (T), tumor margin (M), peritumoral tissue (P) and distant normal skin (N) in cutaneous squamous cell carcinoma (SCC, black bars) and basal cell carcinoma (BCC, white bars). Relative mRNA expression to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and normalized to normal skin is shown. Depicted are mean ± SEM values. *p<0.05, **p<0.01, ***p<0.001 by Kruskal-Wallis test with Dunn’s post-test for comparison between N, P, M and T and by Mann Whitney U test for comparison between SCC and BCC in each group. N = 28 SCC and 19 BCC.
Consistent with the increased expression of TLRs in SCC, gene products that are either downstream of the TLR signaling cascade, or inducible by TLR activation, were also elevated in SCC. TRIF mRNA expression in the SCC tumor center was 17.11±5.14-fold higher than in normal skin (p<0.01), in peritumoral tissue (3.89±2.51, p<0.05) and also significantly higher than in the tumor center of BCCs (3.13±1.36, p<0.001). TRAF1 mRNA expression in the SCC tumor center was 9.62±2.18-fold higher than in normal skin (p<0.01) and also significantly higher than in the tumor center of BCCs (3.40±2.36, p<0.05). TNF mRNA expression in the SCC tumor center was 515±144-fold higher than in normal skin (p<0.001) and also significantly higher than in the tumor center of BCCs (55.5±29.3, p<0.001). In SCC, TNF mRNA expression was also significantly elevated in the tumor margin compared to normal skin (176.2±89.4, p<0.05). BCC showed also significantly higher TNF expression (55.5±29.3 vs. 1.02±0.55, p<0.05) and higher IFNγ expression (17.1±4.1 vs. 1.01±0.04, p<0.05) than in normal skin (
mRNA expression of TIR-domain-containing adaptor-inducing interferon-β (TRIF), TNF receptor-associated factors 1 and 2 (TRAF1 and 2), tumor necrosis factor (TNF) and interferon-γ (IFNγ) in the tumor center (T), tumor margin (M), peritumoral tissue (P) and distant normal skin (N) in cutaneous squamous cell carcinoma (SCC, black bars) and basal cell carcinoma (BCC, white bars). Relative mRNA expression to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and normalized to normal skin is shown. Depicted are mean ± SEM values. *p<0.05, **p<0.01, ***p<0.001 by Kruskal-Wallis test with Dunn’s post-test for comparison between N, P, M and T and by Mann Whitney U test for comparison between SCC and BCC in each group. N = 28 SCC and 19 BCC.
hBD1 mRNA expression in the SCC tumor center was 50.36±28.90-fold higher than in normal skin (p<0.05), in the tumor margin (0.93±0.44, p<0.05) and also significantly higher than in the tumor center of BCCs (1.08±0.45, p<0.001). In BCC hBD1 mRNA expression was significantly lower in the tumor margin than in the tumor center (0.15±0.04 vs. 1.08±0.45, p<0.05). hBD2 mRNA expression in the SCC tumor center was 4748±3934-fold higher than in normal skin (p<0.005), in the tumor margin (5.02±2.11, p<0.01) and also significantly higher than in the tumor center of BCCs (58.36±40.56, p<0.05). In BCC hBD2.
mRNA expression was significantly higher in the tumor center (58.36±40.56) than in the tumor margin (0.61±0.26, p<0.05) or in the peritumoral tissue (0.30±0.11, p<0.01). Psoriasin(S100A7) is an abundant protein in the epidermis with much different antimicrobial activity than hBDs or cathelicidins. At protein concentrations exceeding 100 ug/ml psoriasin can partially inhibit the growth of Gram negative bacteria while hBDs and cathelicidins act at low micromolar concentrations against a broad range of bacteria, viruses and fungi
mRNA expression of cathelicidin antimicrobial peptide (
Protein expression of the antimicrobial peptides hBD1, hBD2 and psoriasin as detected by immunofluorescence staining (green) in normal skin, basal cell carcinoma (BCC) and squamous cell carcinomas in immunocompetent patients (SCC imm.comp.) and organ transplant recipients (SCC OTR). Nuclei were visualized with 4′-6-diamidino-2-phenylindole (DAPI, blue). The top row shows hematoxylin and eosin (HE) staining of the same skin samples. Arrows point at groups of tumor cells. Bar = 100 µm. Data are representative of five samples each.
A direct comparison was made between the expression of all target genes in OTR and immunocompetent patients (ICP). Differences in gene expression between OTR and ICP were only found for antimicrobial peptide genes and are displayed in
mRNA expression of cathelicidin antimicrobial peptide (
As in ICPs, SCC in OTRs showed enhanced hBD2 mRNA expression in the tumor center (233.5±149.2) compared to the tumor margin (3.11±2.16, p<0.05), peritumoral tissue (1.22±0.95, p<0.01) and normal skin (2.14±1.61, p<0.05). However, in contrast to ICPs, SCC in OTRs did not overexpress hBD1 or psoriasin. Psoriasin mRNA expression in the tumor center of SCC in OTRs was significantly lower than in ICPs (71.9±49.1 vs. 1334±690, p<0.01) (
This study sought to provide an in depth analysis of innate immune gene expression in NMSC. The goal of this work was to further understand how our skin responds to the development of tumors and thus gain essential information that can help guide therapy. The candidate genes evaluated represented those such as TLRs that are involved in the innate immune recognition of danger, cytoplasmic molecules such as TRIF and TRAF that transmit information detected by these receptors, and molecules triggered by this process. These last groups of effector molecules are the AMPs and cytokines responsible for mediating the host response once danger is detected. Results show a dramatic difference in expression of many of these critical molecules in SCC, suggesting that this tumor type has enhanced many elements of innate immune response compared to normal skin. Since many of the effector molecules may play a role in the growth of the tumor or resistance to infection, and some are further altered in the immunocompromised host, these may partially explain the natural progression of NMSC.
Although full coverage of all molecules that participate in innate immunity would involve transcriptional profiling of several thousand gene products, and would be best approached by micro-array analysis
Activation of TLRs can result in a wide variety of responses that include apoptosis, inflammatory and non-inflammatory reactions (reviewed in
TRIF and TRAF are downstream signaling molecules of TLR activation and their expression correlated with increased TLR expression in SCC. Similarly, the cytokine response was highest in SCC and correlated with increased TLR expression and signaling. However, in BCC TNF was significantly elevated compared to normal skin despite a lesser TLR response. Increased IFNγ was seen only in BCC. This underlines fundamental differences of innate immune receptor signaling and cytokine expression in BCC and SCC and likely reflects both the local expression of these molecules and expression in recruited inflammatory cells.
Because of the action of AMPs to alter cell growth and differentiation
Psoriasin (S100A7) represents a much different type of molecule than the hBDs or cathelicidin and warrents separate discussion. Psoriasin is a much larger protein, is found in much greater abundance in skin, and has different antimicrobial activity (reviewed in
Immunocompromised patients such as organ transplant recipients (OTRs) have a 60- to 100-fold increased risk for SCC
To investigate if drug-induced immunosuppression altered innate immune gene expression, we included 11 SCC from OTRs for comparison with SCC from immunocompetent patients. All patients in our study were on a treatment regimen with corticosteroids and the mTOR inhibitor rapamycin for at least a year before tumor diagnosis and excision. In epidemiological studies, switching from an immunosuppressive therapy with calcineurin inhibitors to the mTOR-inhibitor sirolimus was effective to reduce skin carcinogenesis in renal transplant patients by 50%
In summary, this study clearly shows that the expression of several genes involved in the innate immune response is different in SCC than normal tissues. There are many possible reasons why skin tumors showed elevated expression of these innate immune genes. Tumor infiltrating immune cells such as CD4+, CD8+ and FOXP3+ T-cells and macrophages secrete soluble effector molecules such as interferons, IL-6 and TNF, and some of these can directly influence innate immune gene expression in keratinocytes (reviewed in
The approach of sampling tissue from the tumor margin and periphery distinguished expression of some genes of interest and provides an example of how basic research might be integrated in well established standardized clinical surgical procedures such as Mohs surgery. Furthermore, it would be a mistake to overlook the remarkably low response seen in BCC for some gene products. The expression (or lack of expression) of unique elements of the innate immune system provides clues to development of the next generation of targeted immune therapy for NMSC.
The study was designed to include 10 – 20 specimens each from SCC in immunocompetent patients, from SCC in organ transplant recipients and from basal cell carcinomas. Following approval by the Human Research Protection Program at the University of California, San Diego (IRB ref. # 071032), and upon receiving written informed consent prior to biopsy, skin specimens from subsequent patients undergoing Mohs surgery for non-melanoma skin cancer at the Division of Dermatology, University of California, San Diego, from December 2009 to June 2010 were collected. The only inclusion criterion was the tumor diagnosis. There were no exclusion criteria. None of the patients in this study was pregnant or had other underlying skin disorders in the areas from which skin samples were obtained. Storage and use of all tissues included in the work presented here was carried out in accordance with the Helsinki declaration. 37 patients were included. 19 patients had a basal cell carcinoma (BCC) and 28 squamous cell carcinoma (SCC). Because immunosuppressed patients such as organ transplant recipients (OTR) have a 60–100-fold greater risk for SCCs this study was designed to include 11 SCCs from OTRs for comparison with 17 SCC from immunocompetent patients (ICP). In the BCC patient group, 68.4% were men, and mean age was 67.1±3.9 years (± SEM). In the SCC ICP group, 76.5% were men and mean age was 74.3±2.7 years. In the SCC OTR group, 81.8% were men. As expected and published before
Mohs microscopically controlled surgery of these skin tumors was performed as described before
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) and 1 µg RNA was reverse transcribed using iScript (BioRad, Hercules, CA). Expression of the cathelicidin gene (
For immunohistofluorescence, frozen sections (6 µm) were fixed with 4% paraformaldehyde, blocked with 3% BSA in phosphate-buffered saline (PBS), and incubated with mouse monoclonal anti-psoriasin antibody (47C1068, sc-52948, Santa Cruz Biotechnology), rabbit anti-hBD1 (FL-68, sc-20797, Santa Cruz) or anti-hBD2 antibody (FL-64, sc-20798, Santa Cruz). Normal mouse IgG (sc-3877, Santa Cruz) and rabbit pre-immune serum were used as negative controls (
Statistical analysis was performed using GraphPad Prism software v5.0 (GraphPad Software Inc., La Jolla). The means and standard errors of the mean (SEM) were calculated for each data set. Data were analyzed by analysis of variance (ANOVA) or the Kruskal-Wallis test with post-tests when appropriate, Mann-Whitney U test and unpaired t test. P-values < 0.05 were considered significant.
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The authors thank Cuong Nguyen for assistance with experiments and Lars E. French for scientific advice.