Modulation of β-catenin /T cell factor activity by Protein Kinase D in SW480 colon cancer cells Free

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Objective: Colorectal cancer is the second-leading cause of cancer-related deaths in the United States. Abnormalities in the expression and functional activity of β-catenin are implicated in the development and progression of colon cancers. We have identified a novel interaction between β-catenin and Protein Kinase D1 (PKD1). Protein Kinase D (PKD) family members (PKD1, 2 and 3) are important modulators of several signal-transduction pathways in benign and malignant human diseases. The main focus of this study is to determine the functions of PKD isoforms in β-catenin signaling in colon cancer.

Materials and Methods: We have selected the human colorectal adenocarcinoma cell line SW480 to study PKD1 mediated effects on β-catenin. PKD1, PKD2 and PKD3 and β-catenin localization was determined by confocal Laser Scanning Microscopy using specific antibodies. The effect of PKD on β-catenin /T cell factor (TCF) transcription activity was measured by luciferase reporter assays. SW480 cells were plated in 96 well plate 16h prior to transient transfection with PKD constructs and the reporter constructsTopflash or Fopflash and pRL-TK (Renilla luciferase; Promega, Madison, WI). Luciferase activities were assayed using the Dual Glo (dual reporter assay, Promega, Madison, WI). Nuclear and membrane targeted PKD1 constructs were made by PCR and cloning. Nuclear and cytoplasmic extracts of SW480-GFP and SW480-GFP-PKD1 cells were prepared using a commercial Nuclear Extract Kit™ (Active Motif, Carlsbad, CA). Expression of β-catenin and PKD1 was studied using a paraffinized human colon cancer tissue micro-array slide.

Results: PKD1 and PKD2 were present at very low levels whereas PKD3 was strongly expressed and localized to nucleus, cytoplasm and cell membranes of SW480 cells. The luciferase assay results showed that PKD1and PKD2 expression leads to reduction in β-catenin /TCF transcriptional activation. There was no significant effect of PKD3 on β-catenin transcription activity. To verify the possible role of PKD1 in modulating β-catenin nuclear expression, we analyzed β-catenin expression in nuclear extracts from GFP-PKD1 and GFP vector expressing SW480 cells. Over expression of PKD1 resulted in decreased nuclear expression of β-catenin in SW480-GFP-PKD1 as compared to parental and vector transfected SW480 cells. Our immunohistochemical study shows down regulation of PKD1 staining in 66 samples that are moderately and poorly differentiated and had a higher Dukes stage (II, III and IV) as compared to non-neoplastic colon samples.

Conclusion: The expression of PKD1 and PKD2 in SW480 colon cancer cells lead to down regulation of β-catenin transcription activity. PKD1 down regulation in colon cancer and its correlation with β-catenin nuclear staining in advanced cancer suggests that PKD1 mediated β-catenin signaling may play a role in carcinogenesis and cancer progression.