Malignant Transformation of Immortalized HaCaT Keratinocytes through Deregulated Nuclear Factor κB Signaling

Abstract

Previous studies addressing functional aspects of nuclear factor KB (NF-KB) activation in normal and transformed keratinocytes revealed complex and seemingly contradictory roles of this transcription factor in this cell type. In normal skin, NF-KB signaling seems to inhibit squamous cell carcinoma development whereas, in squamous cell carcinoma themselves, deregulated NF-KB expression and/or signaling is frequently observed. To further investigate this paradox, we focused on NF-KB activation as it relates to the transformed phenotype of immortalized but nontumorigenic human keratinocytes (HaCaT cells). We observed that NF-KB activity contributed to survival and growth of cultured HaCaT keratinocytes as shown by use of pharmacologic NF-KB inhibitors, RNA interference, and inducible overexpression of a dominant interfering IKB construct. NF-KB activation was largely provided through interaction with extracellular matrix components because preventing cell attachment by forced suspension culture markedly reduced NFKB signaling associated with cell death (anoikis); conversely, anoikis was partially reversed by NF-KB activation induced either by tumor necrosis factor-A treatment or by overexpressing the NF-KB p65 subunit in HaCaT cells. Furthermore, overexpression of NF-KBp65 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-KB signaling supports multiple malignant traits in vitro and in vivo. (Cancer Res 2006; 66(10): 5209-15)

An inactivating CYLD mutation promotes skin tumor progression by conferring enhanced proliferative, survival and angiogenic properties to epidermal cancer cells

“…This could be explained by the higher NF-kB activity found in the C57-CYLD C/S cells, in line with the findings indicating that malignant epidermal cells have accumulated molecular alterations that enable a 'switch' of NFkB function from being a tumor suppressor in normal murine and human keratinocytes (Seitz et al, 1998;van Hogerlinden et al, 1999;Dajee et al, 2003) to being a tumor promoter (Ren et al, 2006). Tumor epidermal cells expressing CYLD C/S also show an important increase in the nuclear localization of Bcl3, p52 and b-catenin.…”

Inhibition of Tumor Promotion by Parthenolide: Epigenetic Modulation of p21

“…The activity of the p65 NF-kB subunit is a limiting factor for NFkB activation and transformation responses (38). Overexpression of p65 in JB6PÀ and HaCaT cells is sufficient for their transformation response to tumor promoters (38,39). Moreover, p65/NF-kB induces the transcription of cyclin D1 (31) and p21 (27) in epidermal cells.…”

Effects of protein phosphorylation on ubiquitination and stability of the translational inhibitor protein 4E-BP1

“…Likewise, the inactivation or loss of 4E-BP1 releases active eIF4E, which stimulates post-transcriptionally the expression of antiapoptotic or oncogenic proteins (Rosenwald et al, 1995;Rousseau et al, 1996; Hoover et al, 1997; Carter Nimmanapalli et al, 2003;Othumpangat et al, 2005b). Since the overactivity or overexpression of either NF-kB or eIF4E inhibits apoptosis and promotes malignant transformation (Topisirovic et al, 2003;Clemens, 2004;Ren et al, 2006), the eIF4E/4E-BP1 system and its regulatory mechanisms can be viewed as a translational parallel to the NF-kB/IkB system.…”