Protein kinase Cε is oncogenic in colon epithelial cells by interaction with the ras signal transduction pathway

Abstract

We have shown previously that overexpression of the e isoform of protein kinase C (PKCe) in rat colonic epithelial cells causes malignant transformation, possibly by interacting with the ras signal transduction pathway (Oncogene 12: 847, 1996). We have now performed experiments to examine certain early steps in the ras signaling pathway. A marked increase of Raf-1 phosphorylation was detected in tumorigenic ras-transformed D/ras as well as in D/e cells (overexpressing PKCe), compared to the nontumorigenic D/WT parental line. Moreover, in the PKCe-transformed D/e cell line, stable transfection with a dominant-negative raf-1 (DNraf) sequence caused complete regression of the neoplastic phenotype. These results suggested that PKCe-induced transformation was associated with increased Raf-1 activation, and that DNraf could block the oncogenic e ect of PKCe. Furthermore, transfection of D/WT cells with dominant-negative ras induced arrest of cell growth, and subsequent transfection with PKCe cDNA enhanced cell proliferation and induced neoplastic transformation. These results suggest that ras acts upstream of PKCe, and that overexpression of PKCe circumvents the block in cell proliferation caused by dominant-negative ras. We conclude that PKCe exerts its oncogenic activity in rat colonic cells by a ecting the ras signaling cascade at the level of Raf-1 activation.Keywords: PKCe; Raf-1; colon cancer; oncogeneWe have demonstrated that the epsilon isoform of protein kinase C (PKCe) is oncogenic when overexpressed in rat colonic epithelial cells . In the previous report, we analysed the expression of several PKC isoforms in a nontumorigenic rat colonic epithelial cell line FRC/TEX CLD (D/WT), and in a derivative tumorigenic Ha-rastransformed FRC/TEX CLD/H-ras line (D/ras) (Pories et al., 1993). PKCe was markedly increased in D/ras cells when compared to the non-neoplastic D/ WT line. To assess whether overexpression of PKCe was involved in the transformed phenotype, we stably transfected D/WT cells with a PKCe cDNA construct. Overexpression of PKCe induced complete in vitro and in vivo neoplastic transformation.Because PKCe expression was found to be altered in D/ras cells, our ®ndings suggested that PKCe might exert its oncogenic activity by interacting with the ras signal transduction pathway. In particular, although it has been shown that p21ras can recruit Raf-1 kinase to the plasma membrane, this interaction is not su cient for complete Raf-1 activation, which occurs via phosphorylation of speci®c serine residues by a Raf kinase-kinase (Daum et al., 1994). Several reports have suggested that PKCe and PKCa are likely candidates for this kinase in mammalian cells (reviewed in: Perletti and Monti, 1996;Daum et al., 1994;Malarkey et al., 1995). We, therefore, focused new experiments on the role of PKCe in ras signal transduction, at the level of Raf-1 activation.Based on the ®ndings described above, we hypothesize that PKCe interacts with the ras signaling pathway, likely by activating Raf-1. Raf-1 phosphorylation is in...

Protein kinase Cε makes the life and death decision

“…Fourth, overexpression of PKCε reversed growth inhibition caused by dominant-negative Ras in NIH 3T3 cells, and rat colonic epithelial cells [41,77]. Expression of dominant-negative Ras had little effect on PKCε-mediated neoplastic transformation [41,76]. These results suggest that PKCε interacts with the Ras signaling pathway acting downstream of Ras but upstream of Raf-1.…”

“…Overexpression of PKCε in NIH 3T3 cells [36] and Rat 6 embryo fibroblasts [35] caused disordered cell growth, such as stimulation in growth rate, increased saturation density and growth in soft agar, and induced tumor formation in nude mice. Overexpression of PKCε was also oncogenic in colonic epithelial cells [41,42]. Although a progressive increase in PKCε was noted in rat liver with increase in malignancy, overexpression of PKCε in rat liver MH1C1 cells did not result in disordered cell growth or tumor formation in nude mice [43].…”

“…Third, introduction of dominant-negative Raf-1 reversed the oncogenic effect of PKCε in Rat 6 fibroblasts and colonic epithelial cells [42,76]. Fourth, overexpression of PKCε reversed growth inhibition caused by dominant-negative Ras in NIH 3T3 cells, and rat colonic epithelial cells [41,77]. Expression of dominant-negative Ras had little effect on PKCε-mediated neoplastic transformation [41,76].…”

Molecular signals in anti-apoptotic survival pathways

“…28,29 PKC and other interacting pathways PKC may also contribute to neoplasia and chemoresistance, through associations with other signalling molecules. Active PKC and Ras co-operate to promote proliferation and transformation, 124,125 however, following PKC inhibition, the pathway stimulated by Ras is apoptosis. 125 Both Ras and PKC enhance Bcl-2 expression levels and anti-apoptotic function.…”

Downregulation of Bid is associated with PKCɛ-mediated TRAIL resistance

“…26 PKCe is the only PKC isoform that has been reported to have oncogenic potential. 27,28 There is also considerable evidence that PKCe acts as an antiapoptotic protein. 23,29,30 Activation of PKC was shown to protect against TRAILinduced apoptosis in several cell lines, including cervical cancer, multiple myeloma, glioma and breast cancer.…”