Differential expression of degradome components in cutaneous squamous cell carcinomas

Abstract

Although the cure rate for cutaneous squamous cell carcinoma is high, the diverse spectrum of squamous cell carcinoma has made it difficult for early diagnosis, particularly the aggressive tumors that are highly associated with mortality. Therefore, molecular markers are needed as an adjunct to current staging methods for diagnosing high-risk lesions, and stratifying those patients with aggressive tumors. To identify such biomarkers, we have examined a comprehensive set of 200 histologically defined squamous cell carcinoma and normal skin samples by using a combination of microarray, QRT-PCR and immunohistochemistry analyses. A characteristic and distinguishable profile including matrix metalloproteinase (MMP) as well as other degradome components was differentially expressed in squamous cell carcinoma compared with normal skin samples. The expression levels of some of these genes including matrix metallopeptidase 1 (MMP1), matrix metallopeptidase 10 (MMP10), parathyroid hormone-like hormone (PTHLH), cyclin-dependent kinase inhibitor 2A (CDKN2A), A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1), FBJ osteosarcoma oncogene (FOS), interleukin 6 (IL6) and reversion-inducing-cysteine-rich protein with kazal motifs (RECK) were significantly differentially expressed (P≤0.02) in squamous cell carcinoma compared with normal skin. Furthermore, based on receiver operating characteristic analyses, the mRNA and protein levels of MMP1 are significantly higher in aggressive tumors compared with non-aggressive tumors. Given that MMPs represent the most prominent family of proteinases associated with tumorigenesis, we believe that they may have an important role in modulating the tumor microenvironment of squamous cell carcinoma.

Figure 1

Microarray analysis of cutaneous squamous cell carcinoma. The gene expression profiles were compared between six tumors and matching normal skin samples. (a) Hierarchical cluster analysis revealing dendrogram and heatmap with distinct gene expression profiles (164 genes with ≥ 5-fold difference) in normal skin and squamous cell carcinoma. Downregulated and upregulated genes are shown in green and red blocks, respectively. (b) The network of the interaction or association between differentially expressed genes. The nodes on the network represent genes/proteins with their biological function (, transcription factor; , generic enzyme; , generic phospholipase; , protein kinase; , generic protease; , metalloprotease; , receptor ligand; , generic receptor; , receptor with enzyme activity; , GPCR; , proteoglycan; , generic-binding protein; , generic channel). Red lines are inhibitory interactions, green lines are activating and gray indicate an unspecified effect in the source literature. Red and blue circles indicate genes upregulated or downregulated, respectively, in tumors compared with normal skin. Note MMP1, MMP10 (stromelysin-2) and ADAM-TS1 are shown in ‘dashed circles’ to indicate the expression pattern and their association with other differentially expressed genes.

Figure 2

Real-time RT-PCR validation of 12 genes (matrix metallopeptidase 1 (MMP1), matrix metallopeptidase 10 (MMP10), parathyroid hormone-like hormone (PTHLH), cyclin-dependent kinase inhibitor 2A (CDKN2A), ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1), FBJ osteosarcoma oncogene (FOS), suppressor of cytokine signaling 3 (SOCS3), thrombospondin 1 (THBS1), interleukin 6 (IL6), reversion-inducing-cysteine-rich protein with kazal motifs (RECK) and complement factor D (CFD)) using 27 paraffin-preserved skin samples. Relative gene expression levels normalized to GAPDH in 22 squamous cell carcinoma and 5 normal skin tissues were determined using gene-specific primers as described in Materials and methods. Gray lines represent medians. Note: as expected from the microarray analysis, MMP1, MMP10, PTHLH, CDKN2A are overexpressed, and ENPP2, ADAMTS1, FOS, SOCS3, THBS1, IL-6, RECK and CFD are underexpressed in squamous cell carcinoma compared with normal skin tissues.

Figure 3

Real-time reverse transcription-polymerase chain reaction (RT-PCR) validation of MMP1, MMP10 and ADAMTS1 using 69 fresh–frozen skin samples. Gene expression levels normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used to differentiate normal skin vs tumors (a) and non-aggressive vs aggressive tumors (b) as described in Materials and methods. Gray lines represent medians.

Figure 4

Immunohistochemistry of matrix metallopeptidase 1 (MMP1) expression in squamous cell carcinoma. High protein expression of MMP1 is detected in aggressive cancer compared with non-aggressive tumors. Note higher proportion of cells stained in aggressive tumor (b) compared with non-aggressive tumors (a); magnification × 200.

Figure 5

Receiver operating characteristic (ROC) analysis of matrix metallopeptidase 1 (MMP1), matrix metallopeptidase 10 (MMP10), A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) expression. The mRNA expression in 69 fresh–frozen samples was tested for the performance of discrimination between: (a) normal skin vs tumors, and (b) non-aggressive vs aggressive tumors. The protein expression in 122 paraffin-preserved samples was also tested for the performance of discrimination between non-aggressive vs aggressive tumors (c). Results are shown for the area under the curve for MMP1 (▲), MMP10 (● and ADAMTS1 (♦). The broken diagonal line denotes an area under the curve = 0.50.