PKCα tumor suppression in the intestine is associated with transcriptional and translational inhibition of cyclin D1

Abstract

Alterations in PKC isozyme expression and aberrant induction of cyclin D1 are early events in intestinal tumorigenesis. Previous studies have identified cyclin D1 as a major target in the antiproliferative effects of PKCα in non-transformed intestinal cells; however, a link between PKC signaling and cyclin D1 in colon cancer remained to be established. The current study further characterized PKC isozyme expression in intestinal neoplasms and explored the consequences of restoring PKCα or PKCδ in a panel of colon carcinoma cell lines. Consistent with patterns of PKC expression in primary tumors, PKCα and δ levels were generally reduced in colon carcinoma cell lines, PKCβII was elevated and PKCε showed variable expression, thus establishing the suitability of these models for analysis of PKC signaling. While colon cancer cells were insensitive to the effects of PKC agonists on cyclin D1 levels, restoration of PKCα downregulated cyclin D1 by two independent mechanisms. PKCα expression consistently (a) reduced steady-state levels of cyclin D1 by a novel transcriptional mechanism not previously seen in non-transformed cells, and (b) re-established the ability of PKC agonists to activate the translational repressor 4E-BP1 and inhibit cyclin D1 translation. In contrast, PKCδ had modest and variable effects on cyclin D1 steady state levels and failed to restore responsiveness to PKC agonists. Notably, PKCα expression blocked anchorage-independent growth in colon cancer cells via a mechanism partially dependent on cyclin D1 deficiency, while PKCδ had only minor effects. Loss of PKCα and effects of its re-expression were independent of the status of the APC/β-catenin signaling pathway or known genetic alterations, indicating that they are a general characteristic of colon tumors. Thus, PKCα is a potent negative regulator of cyclin D1 expression and anchorage-independent cell growth in colon tumor cells, findings that offer important perspectives on the frequent loss of this isozyme during intestinal carcinogenesis.

MiniSOX9, a dominant-negative variant in colon cancer cells

“…Furthermore, we evidenced that MiniSOX9 accumulates in the nucleus, inhibits SOX9 DNA-binding-dependent transcriptional activity, inhibits PKCa expression and stimulates the canonical Wnt signaling pathway, known to activate in colon cancers. All these features call for an oncogenic role of MiniSOX9, because PKCa behaves as a tumor suppressor in the intestine epithelium (Pysz et al, 2009) and the tumor-promoting activity of Wnt/b-catenin is well documented (MacDonald et al, 2009). How MiniSOX9, devoid of transactivation domain, can activate Wnt?…”

Heat Shock Proteins Regulate Activation-induced Proteasomal Degradation of the Mature Phosphorylated Form of Protein Kinase C

“…Adenoviral Overexpression of PKC␣-IEC-18 cells were infected with PKC␣ adenovirus at a multiplicity of infection of 5 for 16 h in IEC-18 minimal medium as follows: DMEM supplemented with 2% FBS, 4 mM L-glutamine, and 5 g/ml insulin (62). The medium was then replaced with IEC-18 complete medium, and cells were subjected to the indicated treatments after an additional 24 -48 h.…”

“…Immunoblot Analysis-Cells were lysed with 1% SDS, which extracts PKCs from the cytosol, membrane, and nonionic detergent-insoluble fractions, and immunoblot analysis of whole cell lysates was performed as we have described (28,62) using SuperSignal West Pico ECL (ThermoScientific). The apparent molecular weight of bands was determined based on electrophoretic migration relative to prestained standard proteins (Bio-Rad/ThermoScientific).…”

Protein Kinase C (PKC) Isozymes and Cancer

“…Although the levels of PKC are similar between normal and cancerous colon tissue, increasing evidence shows that PKC does participate in growth arrest [107,108] and cancer suppression in the intestine epithelium [109][110][111][112][113]. Suppression of cell growth in colon cancer cells by PKC is thought to occur through the regulation of EGFR signaling [110]; ERKmediated inhibition of the inhibitor of DNA binding 1, a proproliferative factor [112]; downregulation of -catenin and its target genes cyclin D1 and c-Myc [111,113]; and PKCmediated phosphorylation of retinoic acid-related orphan nuclear receptor , which downregulates the Wnt/ -catenin signaling [114].…”