Repression of Protein Kinase C Delta in Human Squamous Cell Carcinomas by Ras, Fyn and NF-Kappa B Signaling
The delta isoform of Protein Kinase C (PKC-delta) is widely expressed in many normal tissues, including epidermal keratinocytes, and has a critical role in UV-induced apoptosis. However, PKC-delta is frequently lost in chemically or UV-induced mouse skin tumors, as well as in human cutaneous squamous cell carcinomas (SCC). Furthermore, re-expression of PKC-delta in human SCC lines is sufficient to induce apoptosis and suppress tumorigenicity, making PKC-delta a potential tumor suppressor gene for SCCs. The objective of this dissertation is to investigate the mechanism of PKC-delta loss in human SCCs. To determine the mechanism of PKC-delta loss in human SCCs, we used Laser Capture Microdissection to isolate cells for RNA and DNA analysis from 3 normal epidermises and 14 human SCCs with low PKC-delta protein. Using this more selective approach, we found the tumor suppressor PKC-delta is lost at the mRNA level in human SCCs, and that the PKC-delta gene is rarely deleted suggesting that the mechanism of down-regulation of PKC-delta in SCCs is likely to be primarily at the level of gene transcription. To further explore the mechanism of PKC-delta down-regulation, we studied Ras-transformed immortalized human keratinocytes (HaCaT-Ras), which have selective down-regulation of the PKC-delta isoform at both protein and mRNA levels. Ras significantly repressed human PKC-delta promoter activity in HaCaT cells (85% reduction, p<0.05). Mutagenesis and ChIP studies of the PKC-delta promoter revealed that Ras activation represses PKC-delta promoter activity by activation of nuclear factor kappa B (NF-kB) and recruitment of repressive NF-kB subunits (p50 and c-Rel). We also found that Fyn tyrosine kinase activation was necessary and sufficient for NF-kB activation and PKC-delta repression. In addition, Fyn was over-expressed in human SCCs and HaCaT-Ras cells. Furthermore, activation of PI3K/AKT pathway was necessary and sufficient for Ras-induced up-regulation of Fyn expression in HaCaT cells. Thus, a Ras-PI3K-Fyn-NF-kB pathway leads to PKC-delta repression in human keratinocytes. Our results have implications for the development of therapeutic strategies abrogating this signaling pathway to trigger the re-expression of pro-apoptotic PKC-delta to induce apoptosis in SCCs.
fyn, nuclear factor kappaB, pkc delta, ras, skin cancer, tumor suppressor, Molecular Biology
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