Protein Kinase C Delta Negatively Modulates Canonical Wnt Pathway and Cell Proliferation in Colon Tumor Cell Lines

PLoS One. 2013;8(3):e58540. doi: 10.1371/journal.pone.0058540. Epub 2013 Mar 8.

Abstract

The tumor suppressor Adenomatous Polyposis coli (APC) gene is mutated or lost in most colon cancers. Alterations in Protein kinase C (PKC) isozyme expression and aberrant regulation also comprise early events in intestinal carcinomas. Here we show that PKCδ expression levels are decreased in colon tumor cell lines with respect to non-malignant cells. Reciprocal co-immunoprecipitation and immunofluorescence studies revealed that PKCδ interacts specifically with both full-length (from non-malignant cells) and truncated APC protein (from cancerous cells) at the cytoplasm and at the cell nucleus. Selective inhibition of PKCδ in cancer SW480 cells, which do not possess a functional β-catenin destruction complex, did not affect β-catenin-mediated transcriptional activity. However, in human colon carcinoma RKO cells, which have a normal β-catenin destruction complex, negatively affected β-catenin-mediated transcriptional activity, cell proliferation, and the expression of Wnt target genes C-MYC and CYCLIN D1. These negative effects were confirmed by siRNA-mediated knockdown of PKCδ and by the expression of a dominant negative form of PKCδ in RKO cells. Remarkably, the PKCδ stably depleted cells exhibited augmented tumorigenic activity in grafted mice. We show that PKCδ functions in a mechanism that involves regulation of β-catenin degradation, because PKCδ inhibition induces β-catenin stabilization at the cytoplasm and its nuclear presence at the C-MYC enhancer even without Wnt3a stimulation. In addition, expression of a dominant form of PKCδ diminished APC phosphorylation in intact cells, suggesting that PKCδ may modulate canonical Wnt activation negatively through APC phosphorylation.

Publication types

  • Clinical Trial
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenomatous Polyposis Coli Protein / genetics
  • Adenomatous Polyposis Coli Protein / metabolism*
  • Animals
  • Cell Line, Tumor
  • Cell Nucleus / genetics
  • Cell Nucleus / metabolism
  • Cell Nucleus / pathology
  • Cell Proliferation*
  • Colonic Neoplasms / metabolism*
  • Colonic Neoplasms / pathology
  • Cyclin D1 / genetics
  • Cyclin D1 / metabolism
  • Cytoplasm / genetics
  • Cytoplasm / metabolism
  • Enhancer Elements, Genetic / genetics
  • Humans
  • Mice
  • Mice, Nude
  • Phosphorylation / genetics
  • Protein Kinase C-delta / genetics
  • Protein Kinase C-delta / metabolism*
  • Protein Stability
  • Proteolysis
  • Proto-Oncogene Proteins c-myc / genetics
  • Proto-Oncogene Proteins c-myc / metabolism
  • Wnt Signaling Pathway*
  • Wnt3A Protein / genetics
  • Wnt3A Protein / metabolism*
  • beta Catenin / genetics
  • beta Catenin / metabolism

Substances

  • APC protein, human
  • Adenomatous Polyposis Coli Protein
  • CCND1 protein, human
  • MYC protein, human
  • Proto-Oncogene Proteins c-myc
  • WNT3A protein, human
  • Wnt3A Protein
  • beta Catenin
  • Cyclin D1
  • PRKCD protein, human
  • Protein Kinase C-delta

Grant support

This research was supported by grants from the Universidad Nacional Autónoma de México (DGAPA-UNAM IN226111) to MRF and from CONACYT (151731) to MRF. JGHM was supported by a doctoral fellowship from CONACYT, Mexico. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.