Given the associations between chronic inflammation and epithelial cancer, we studied susceptibility to skin carcinogenesis in mice deficient for the pro-inflammatory cytokine TNF-alpha (refs. 5,6). TNF-alpha(-/-) mice were resistant to development of benign and malignant skin tumors, whether induced by initiation with DMBA and promotion with TPA or by repeated dosing with DMBA. TNF-alpha(-/-) mice developed 5-10% the number of tumors developed by wild-type mice during initiation/promotion and 25% of those in wild-type mice after repeated carcinogen treatment. TNF-alpha could influence tumor and stromal cells during tumor development. The early stages of TPA promotion are characterized by keratinocyte hyperproliferation and inflammation. These were diminished in TNF-alpha(-/-) mice. TNF-alpha was extensively induced in the epidermis, but not the dermis, in TPA-treated wild-type skin, indicating that dermal inflammation is controlled by keratinocyte TNF-alpha production. Deletion of a TNF-alpha inducible chemokine also conferred some resistance to skin tumor development. TNF-alpha has little influence on later stages of carcinogenesis, as tumors in wild-type and TNF-alpha(-/-) mice had similar rates of malignant progression. These data provide evidence that a pro-inflammatory cytokine is required for de novo carcinogenesis and that TNF-alpha is important to the early stages of tumor promotion. Strategies that neutralize TNF-alpha production may be useful in cancer treatment and prevention.

Keratinocyte-derived TNF-a acts as an endogenous tumour promoter and can also regulate AP-1 activity in mouse epidermis. To gain further insight into TNF-a signalling during skin tumour formation, mice deficient in TNFR1 (TNFR1 À/À mice) or TNFR2 (TNFR2 À/À mice) were subjected to chemical carcinogenesis. Tumour multiplicity was significantly reduced in TNFR1 À/À and TNFR2 À/À mice compared to wild-type (wt) mice, suggesting that both receptors have protumour activity. However, TNFR1 À/À mice were markedly more resistant to tumour development than TNFR2 À/À mice indicating that TNFR1 is the major mediator of TNF-a-induced tumour formation. TNFR1 and TNFR2 were both expressed in wt epidermis during tumour promotion and by primary keratinocytes in vitro. TPA-induced c-Jun expression was transient in TNFR1 À/À and TNFR2 À/À compared to wt epidermis and this was reflected by reduced induction of the AP-1-responsive genes granulocyte/macrophage-colony stimulating factor, matrix metalloproteinase-9 and matrix metalloproteinase-3. These genes were differentially regulated in TNFR1 À/À compared to TNFR2 À/À epidermis, suggesting that the TNF-a receptors act independently via different AP-1 complexes to transduce TNF-a signals during tumour promotion. In addition, TNFR2 cooperated with TNFR1 to optimise TNFR1-mediated TNF-a bioactivity on keratinocytes in vitro. Our data provide further insight into TNF-a signalling in malignancy and provide some rationale for the use of TNF-a antagonists in the treatment of cancer.

Mice deficient in TNF-a (TNF-a À/À mice) are resistant to skin carcinogenesis and expression of MMP-9 is inhibited in TNF-a À/À mice during skin tumour development. In the early stages of tumour promotion, MMP-9 protein initially localized to the follicular epidermis but subsequently began to accumulate in the interfollicular epidermis of wild-type but not TNF-a À/À mice. Inhibition of TNF-a or MMP-9 function reduced keratinocyte migration in vitro. In addition, a deficiency of TNF-a delayed re-epithelialization in vivo and this correlated with reduced MMP-9 expression. Collectively, these data suggest that MMP-9 regulates keratinocyte migration in a TNF-a-dependent manner. Expression profiling of genes that control cell adhesion and migration revealed markedly lower levels of the integrin subunits av and b6 in TNF-a À/À compared with wild-type keratinocytes in vitro. avb6 expression was upregulated by keratinocytes in vitro and during tumour promotion in vivo in a TNF-adependent manner. Furthermore, avb6 blockade significantly inhibited keratinocyte migration and TNFa-stimulated MMP-9 expression in vitro. These data illustrate a novel TNF-a-dependent mechanism for the control of avb6 expression and suggest one pathway for TNF-a regulation of MMP-9. Increased MMP-9 and avb6 expression may stimulate epithelial cell migration during tumour formation and may be one mechanism whereby TNF-a acts as an endogenous tumour promoter.

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The protein-tyrosine phosphatase PTP␣ has been proposed to play an important role in controlling the dephosphorylation of a number of key signaling proteins and in regulating insulin signaling. To examine the potential cellular functions and physiological substrates of PTP␣, a potent phosphorothioate oligonucleotide-based antisense strategy was developed that specifically depleted endogenous PTP␣ from 3T3-L1 adipocytes. The antisense probe, ␣AS1, achieved PTP␣ depletion levels normally of >85% and which varied up to levels where PTP␣ was not detected at all. Elimination of PTP␣ by 85% inhibited c-Src activity by 80%. Abolishing PTP␣ to levels undetected did not alter the tyrosine dephosphorylation of the insulin receptor or insulin receptor substrate proteins. Moreover, the ability of insulin to activate ERK2 or to stimulate DNA synthesis was not altered by ␣AS1. It is concluded that endogenous PTP␣ is a key regulator of c-Src activity in 3T3-L1 adipocytes and that PTP␣ is not required for the dephosphorylation of the insulin receptor or the insulin receptor substrate proteins or for the regulation of several downstream insulin signaling events in 3T3-L1 adipocytes. Finally, the development of the antisense probe, ␣AS1, provides an important molecular tool of general applicability for further dissecting the roles and precise targets of endogenous PTP␣.