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.
Highlights
Colorectal cancer (CRC) is one of the most prevalent cancers and is a leading cause of cancer mortality worldwide
Adenomatous polyposis coli (APC) is Phosphorylated by PKCd in Intact RKO Cells Given that it has been demonstrated that phosphorylation is an important mechanism of APC regulation for modulating its affinity for b-catenin, we examined whether APC could be a PKCd substrate
Many studies have implicated Protein kinase C (PKC) isoforms in Wnt signaling, in non-canonical Wnt activation, the molecular mechanisms involved in their crosstalk remain poorly understood
Summary
Colorectal cancer (CRC) is one of the most prevalent cancers and is a leading cause of cancer mortality worldwide. A great amount of experimental evidence has shown that APC is the gatekeeper in the molecular pathogenesis of the majority of sporadic and hereditary forms of colorectal carcinoma [1,2]. In the adenoma-carcinoma sequence of sporadic colorectal carcinoma (CRC), the smallest identifiable lesion is an aberrant crypt focus (ACF) and two types of ACFs have been distinguished. The second type, the dysplastic ACF or unicryptal adenoma, occurs frequently in carcinoma-associated colon mucosa. Most of these dysplastic ACFs bear APC mutations, whereas nonmalignant hyperplastic ACFs may arise from activating mutations in K-RAS or complementary mutations in the upstream component B-RAF [3,4]
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