DSG3 silencing increased the interaction of plakoglobin with the transcriptional factor TCF.

<p>(<b>A, B</b>) Knockdown of DSG3 increased plakoglobin binding to the TCF4 transcriptional factor, as determined by immunoprecipitation (IP) and immunoblot (IB) analysis as determined in OECM1 (A) and SAS (B) cells. Cellular extracts of the vector or the shDSG3 stable transfected cells were immunoprecipitated with anti-TCF4, rabbit IgG (IgG) (as a negative control) and subsequently immunoblotted with plakoglobin or β-catenin antibody. (<b>C</b>) Immunofluorescence and confocal microscopy were used to examine the interactions among plakpglobin and TCF4. The vector or shDGS3 stable transfected cells were co-stained with either plakoglobin and TCF4 antibodies. After washing, the slides were incubated with rhodamine- or FITC- conjugated second antibody. DAPI staining was performed for nuclear staining. The scale bar indicates the size as 40 µm.</p

Hypothetical model illustrating the DSG3-plakoglobin signaling pathway modulating cell carcinogenesis in head and neck cancer.

<p>(A) In cancer cells, DSG3 is overexpressed and captures more molecules of plakoglobins in the membrane. Loss of the antagonist function of plakoglobin results in more molecules of β-catenin entering the nucleus, which subsequently activates the expression of TCF/LEF downstream target genes c-myc, cyclin D1, and MMP-7 resulting in malignant cells. (B) Silencing DSG3 by shRNA treatment reverses the effects of DSG3, resulting in translocation of plakoglobin to the nucleus thus rendering the negative regulatory effect of plakoglobin on TCF/LEF transcription. (Pg: plakoglobin; B: β-catenin).</p

DSG3 silencing suppresses the TCF/LEF transcriptional activity and the downstream target genes c-myc, cyclin D1, and MMP-7.

<p>(<b>A</b>) and (<b>B</b>) The effects of TCF/LEF transcriptional activity after Plakoglobin or DSG3 knockdown. The stable plakoglobin silencing (sh-Pg) or DSG3 silencing (sh-D3) cells were transfected with TOPflash or FOPflash luciferase reporter plasmid and the Renilla plasmid. After 24 hours, the luciferase activity was determined using the Steady-Glo Luciferase Reagent. The firefly luciferase activity was normalized against the Renilla luciferase activity and the fold increase of the TOPflash activity compared to the FOPflash activity was reported. (<b>C</b>) and (<b>D</b>) The expressions of the TCF/LEF downstream target genes c-myc and cyclin D1 was determined in the cells stably transfected with specific shRNAs target to plakoglobin (sh-Pg) or DSG3 (sh-D3) cells, compared to the vector transfected stably cells. In each sample, proteins were extracted and analyzed by western blot assays to determine the expression levels of c-myc, cyclin D1, and MMP-7.</p

Elevation of DSG3 is associated with plakoglobin translocation in cancer tissues.

<p>(<b>A</b>) Cells from two clones of normal keratinocytes (HNK1, HNK2) and two cancer cell lines (OECM1, SAS) were examined. (B) Clinical tissue samples of four grossly normal biopsies and five pieces of cancer tissues from head-neck cancer patients were examined. Cellular or nuclear proteins were extracted and subjected to immunoblot analysis for the expression of DSG3, plakoglobin, c-myc, Cyclin D1 and MMP7, as described in the method section. Expressions of Actin served as internal control for cellular protein, and HDAC for nuclear protein. Cellular protein expression was normalized by Actin and nuclear protein by HDAC after determination of each band density by Image J software.</p

DSG3 silencing suppressed the growth of xenografted tumors, which is associated with plakoglobin translocation and reduced expressions of TCF target genes.

<p>(<b>A</b>) Six of each group BALB/C null mice were subcutaneously injected with 5×10⁵ cells of the SAS cell line stably transfected with the vector (V) or the shDSG3 (sh2) plasmids. The tumor size was measured every 4 days starting two weeks after injection. Each dimension of the tumors were measured by calipers, and the tumor size was calculated as length×width×height. (***: p<0.001). (<b>B</b>) Five weeks after tumor grafting, the tumors were removed subjected to western blot analysis for the expression of c-myc, cyclin D1, and MMP-7 proteins. (<b>C</b>) The grafted tumors were subjected to immunohistochemistry staining for the localization and expression of plakoglobin, c-myc, cyclin D1, and MMP-7 proteins.</p