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Activation of WNT Family Expression and Signaling in Squamous Cell Carcinomas of the Oral Cavity

¹ Department of Oral Surgery, Graduate School of Dentistry, and
² Department of Biochemistry, School of Dentistry at Tokyo, The Nippon Dental University, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan;

View larger version (33K): [in a new window]   Figure 1. Expression of WNT family members in oral squamous carcinoma cell lines. (A) A panel of WNT members amplified by RT-PCR is listed in the Table . Normal keratinocytes (NKC1 and NKC2) and fibroblasts (GF12) isolated from gingiva were included. (B) Representative data of RT-PCR for WNT3, WNT6, and GAPDH as an internal control (lane 1, OSC19; lane 2, HSC2; lane 3, TSU; lane 4, HOC313; lane 5, Ca9.22; lane 6, KOSC3; lane 7, Ho1u1; lane 8, SCCTF; lane 9, SCCKN; lane 10, HSC3; lane 11, KOSC2). RNA sample isolated from normal gingival keratinocytes (lane 12, NKC1; lane 13, NKC2) and GF12 normal gingival fibroblasts (lane 14) were also applied. PCR reactions without reverse transcription of OSC19 cells were also performed as a negative control (-RT).

View larger version (43K): [in a new window]   Figure 2. Activation of the WNT signaling and downstream target genes. (A) Immunocytochemistry of WNT3 (a,d), GSK3B (b,e), and CTNNB1 (c,f) in carcinoma cells. WNT-negative HSC2 cells (a-c) and WNT-expressing TSU cells (d-f) cultured on glass slides were applied for fluorescent immunostaining. Bar = 25 µm. (B) Analysis of WNT signaling activation in carcinoma cells. Total cellular lysates or nuclear extracts from HOC313 (lane 1), TSU (lane 2), HSC2 (lane 3), or SCCKN (lane 4) were subjected to Western blot. (C) Expression of WNT target genes in carcinoma cells. Size-fractionated protein of HOC313 (lane 1), TSU (lane 2), HSC2 (lane 3), or SCCKN (lane 4) was reacted to specific antibody to c-MYC or MT1-MMP. c-MYC was localized to the nuclei of TSU cells (b), but not to HSC2 cells (a). Bar = 25 µm.

View larger version (105K): [in a new window]   Figure 3. Immunolocalization of WNT3 or CTNNB1 in oral carcinoma tissue. (A) WNT3 was localized to carcinoma cells at the invasive front. (B) Endothelial cells (small arrow), fibroblast-like cells (arrowhead), and macrophage-like cells (large arrow) adjacent to carcinoma cells were also positively stained. (C) Normal gingiva did not react to WNT3 antibody. (D,E) CTNNB1 stained cell-cell junction of carcinoma cells. (F,G) At the extremity of carcinoma invasion, CTNNB1 showed diffuse cytoplasmic or nuclear staining. Bar = 100 µm (A,C–E) and 50 µm (B,F,G).