Malignant transformation of immortalized HaCaT keratinocytes through deregulated nuclear factor κB signaling

Previous studies addressing functional aspects of nuclear factor κB (NF-κB) activation in normal and transformed keratinocytes revealed complex and seemingly contradictory roles of this transcription factor in this cell type. In normal skin, NF-κB signaling seems to inhibit squamous cell carcinoma development whereas, in squamous cell carcinoma themselves, deregulated NF-κB expression and/or signaling is frequently observed. To further investigate this paradox, we focused on NF-κB activation as it relates to the transformed phenotype of immortalized but nontumorigenic human keratinocytes (HaCaT cells). We observed that NF-κB activity contributed to survival and growth of cultured HaCaT keratinocytes as shown by use of pharmacologic NF-κB inhibitors, BNA interference, and inducible overexpression of a dominant interfering IκB construct. NF-κB activation was largely provided through interaction with extracellular matrix components because preventing cell attachment by forced suspension culture markedly reduced NFκB signaling associated with cell death (anoikis); conversely, anoikis was partially reversed by NF-κB activation induced either by tumor necrosis factor-α treatment or by overexpressing the NF-κB p65 subunit in HaCaT cells. Furthermore, overexpression of NF-κBp65 in HaCaT cells induced colony formation in soft agar and tumorigenicity in nude mice. In summary, as opposed to normal keratinocytes, immortalized HaCaT keratinocytes provide a cellular context in which deregulated NF-κB signaling supports multiple malignant traits in vitro and in vivo.