Genomic Analysis Defines a Cancer-Specific Gene Expression Signature for Human Squamous Cell Carcinoma and Distinguishes Malignant Hyperproliferation from Benign Hyperplasia

Abstract

Using high-density oligonucleotide arrays, we measured expression of >12,000 genes in surgical excisions of invasive human squamous cell carcinomas (SCCs) versus site-matched control skin. This analysis defined >1,900 genes with altered expression in SCCs that were statistically different from controls. As SCCs are composed of epithelial cells, which are both hyperplastic and invasive, we sought to define gene sets associated with these biologic processes by comparing gene expression to psoriasis vulgaris, which is a condition of benign keratinocyte hyperplasia without invasiveness or pre-malignant potential. Through this analysis, we found genes that were commonly upregulated in both conditions and unique genes with increased expression in SCCs. Differential gene regulation in these two conditions was confirmed by real-time reverse transcription-PCR and immunohistochemistry. We found that benign hyperplasia is associated with upregulation of genes including DEFB4 (defensin B4), SERPINB3 (serine proteinase inhibitor, member 3), STAT1 (signal transducer and activator of transcription 1), K16 (keratin 16), CEACAMs (carcinoembryonic antigen-related cell adhesion molecules), and WNT 5A (wingless-type MMTV integration site family, member 5A). WNT receptor frizzled homolog 6 (FZD6) and prostaglandin-metabolizing enzyme hydroxyprostaglandin dehydrogenase were increased in SCC alone. Growth factor pleiotrophin (PTN) was expressed at higher levels in non-tumor-bearing skin adjacent to excised SCC. SCC was further characterized by upregulation of matrix metalloproteinases 1, 10, and 13, cathepsin L2, cystatin E/M as well as STAT3 and microseminoprotein, beta (MSMB), and downregulation of inducible nitric oxide synthase, granzyme B, CD8, and CD83. The current study defines a unique gene expression signature for cutaneous SCC in humans and suggests potential roles for WNT, FZD, and PTN in the pathogenesis of SCC.

Molecular discrimination of cutaneous squamous cell carcinoma from actinic keratosis and normal skin

“…These genes have also been found to be consistently overexpressed in other microarray and RT-PCR studies comparing cutaneous squamous cell carcinoma with normal skin. 7,8,56 WNT5a and MMP1 have also been found to be overexpressed in squamous cell carcinoma from other anatomic sites, including the lip, oral cavity, head and neck, and lung. WNT5A is a member of the Wnt family of proteins (discussed previously) and was found to be significantly overexpressed in squamous cell carcinoma in comparison with normal skin in our study (FC ¼ 4.35; Table 2).…”

“…WNT5a was found to be overexpressed in another microarray study comparing cutaneous squamous cell carcinoma with normal skin. 8 Upregulation of WNT5a appears conserved in squamous cell carcinomas from different anatomic locations, including the lip, head and neck, and lung, and appears to be an important gene in the tumorigenesis of squamous cell carcinoma. [56][57][58] Upregulation of WNT5a has been identified in many different malignancies, including melanoma and carcinomas from the breast, esophagus, lung, pancreas, and stomach.…”

“…65 MMP1 is one of the rare genes consistently overexpressed in the different microarray studies examining cutaneous squamous cell carcinomas in comparison with normal skin. 7,8 Upregulation of MMP1 appears to a conserved in squamous cell carcinomas from different anatomic locations including the head and neck and oral cavity. [67][68][69][70] Matrix metallopeptidases appear integral to tumorigenesis and have been targeted by inhibitors; however, clinical trials of advanced lung, prostate, pancreas, brain, and GI tract malignancies have shown little efficacy to date.…”

“…[5][6][7][8] These discrepancies from previous studies are likely due to the type of tissues analyzed (cell line vs fresh tissue vs formalin-fixed and paraffin-embedded tissue) and advances in microarray technology including increasingly sophisticated RNA isolation techniques from formalinfixed and paraffin-embedded tissue. We also utilized the most comprehensive microarray (Affymetrix U133plus2.0 array) to date that examined over 47 000 genes.…”

“…[5][6][7][8] These studies have been hampered by limited sample sizes, poor reproducibility, and the use of cell lines. [5][6][7][8] We profiled and examined the gene expression of over 47 000 genes using the most comprehensive GeneChip microarrays available (human U133 plus 2.0 array), and identified differentially expressed genes among normal skin, actinic keratosis, and squamous cell carcinoma using formalin-fixed and paraffin-embedded tissue. Our goal was to identify unique gene signatures that can discriminate between normal skin, actinic keratosis, and squamous cell carcinoma, and examine the molecular pathways of carcinogenesis involved in the progression from normal skin to squamous cell carcinoma.…”