TNF-alpha Drives Matrix Metalloproteinase-9 in Squamous Oral Carcinogenesis

Objectives/hypothesis: It is well known that invasion is a seminal event in the progression of oral and other head and neck carcinoma sites. We have previously demonstrated tumor necrosis factor (TNF)-alpha and its dependent cytokines are upregulated in saliva during oral carcinogenesis. TNF-dependent events stimulate nuclear factor (NF)-kappaB and many NF-kappaB-dependent genes are associated with cancer progression.

Materials And Methods: In the present study, we examined NF-kappaB stimulation of matrix metalloproteinase (MMP)-9 in a precancerous keratinocyte cell line that models leukoplakia (Rhek cells). We stimulated Rhek cells with both TNF-alpha and phorbol myristate acetate, known stimulants of NF-kappaB. We then assayed MMP-9 transcription and secretion by luciferase reporter genes, quantitative real-time polymerase chain reaction, and fluorometric enzyme-linked immunosorbent serologic assay.

Results: We discovered that the MMP-9 promoter was significantly stimulated by phorbol myristate acetate and TNF-alpha on luciferase reporter gene assays. Further, we uncovered that functional MMP-9 promoter activation was accompanied by significant increases in MMP-9 gene expression, as judged by quantitative real-time polymerase chain reaction. Functional activation of the MMP-9 protein was stimulated by TNF-alpha and PMA on a fluorescent enzyme-linked immunosorbent serologic assay. Finally, we searched our salivary proteomic database for increases in MMP-9 and discovered it was the third most significant protein in salivas of oral cavity cancer patients over normal controls.

Conclusions: We conclude the milieu cytokine, TNF-alpha, has the capacity to provide stimulation of events related to early invasion of oral cavity cancer, as judged by its ability to stimulate MMP-9.