Expression and regulation of the ΔN and TAp63 isoforms in salivary gland tumorigenesis clinical and experimental findings

Abstract

The TP63 gene, a TP53 homologue, encodes for two main isoforms by different promoters: one retains (TA) and the other lacks (ΔN) the transactivation domain. p63 plays a critical role in the maintenance of basal and myoepithelial cells in ectodermally derived tissues and is implicated in tumorigenesis of several neoplastic entities. However, the biological and regulatory roles of these isoforms in salivary gland tumorigenesis remain unknown. Our results show a reciprocal expression between TA and ΔN isoforms in both benign and malignant salivary tumors. The most dominantly expressed were the ΔN isoforms, whereas the TA isoforms showed generally low levels of expression, except in a few tumors. High ΔNp63 expression characterized tumors with aggressive behavior, whereas tumors with high TAp63 expression were significantly smaller and less aggressive. In salivary gland cells, high expression of ΔNp63 led to enhanced cell migration and invasion and suppression of cell senescence independent of TAp63 and/or TP53 gene status. We conclude the following: i) overexpression of ΔNp63 contributes to salivary tumorigenesis, ii) ΔNp63 plays a dominant negative effect on the TA isoform in the modulation of cell migration and invasion, and iii) the ΔN isoform plays an oncogenic role and may represent an attractive target for therapeutic intervention in patients with salivary carcinomas.

Figure 1

Expression of the p63 isoforms in salivary gland tumors. A: The schematic of the TP63 gene. Arrows indicate the isoform-specific primers. B: Expression of p63 isoforms in benign tumors. WT indicates Warthin's tumor; ME, myoepithelioma. C: Expression of p63 isoforms in malignant tumors. AdCC indicates adenoid cystic carcinoma; MYC, myoepithelial carcinoma; PLGA, polymorphous low-grade adenocarcinoma; Aci, acinic cell carcinoma; SDC, salivary duct carcinoma. D: Expression levels of TAp63 and ΔNp63 in the tumors and normal salivary glands by qPCR. The asterisk shows that TA63 and ΔNp63 were detected by RT-PCR. E: Western blotting analysis of p63 isoforms using anti–p63 4A4.

Figure 2

Photomicrographs of an H&E-stained adenoid cystic carcinoma (A) and MEC (case 58) (C) with corresponding p63 immunostaining (B and D). There was restricted expression of p63 staining to the periductal myoepithelial cells in AdCC (B) and diffuse staining in basal and epidermoid cells of case 58 MEC (D).

Figure 3

Expression of p63 isoforms in salivary gland cell lines and inhibition of p63 by siRNA. A: Expression level of p63 isoforms by RT-PCR, Western blot, and qPCR. B: A253 cells were transfected with the siRNA oligonucleotide of all p63-specific (p63), ΔNp63 isoform–specific (ΔNp63), TAp63 isoform–specific (TAp63), or a negative control siRNA (control). Knockdown of each p63 isoform by siRNA revealed that TAp63 expression was induced by ΔNp63-specific siRNA in A253 cells.

Figure 4

Inhibition of p63 by siRNA affected cellular motility, invasion, proliferation, and senescence. A: The invasion assay showed that all p63 isoform (p63) and ΔNp63 siRNAs inhibited the motility of A253 cells compared with control siRNA. A253 cells transfected with a negative control, p63, ΔNp63, or TAp63 siRNA for 48 hours were seeded and incubated in Boyden chambers. Invasive cells were counted after 48 and 72 hours in three independent experiments. B: A would-healing assay showed that the TAp63 knockdown by siRNA led to increased cell motility. A253 cells transfected with a negative control, p63, ΔNp63, or TAp63 siRNA for 48 hours were scratched (0 hours, top) and observed after 24 hours (bottom). C: Knockdown of ΔNp63 or total p63 led to reduced cell proliferation with no effect on apoptosis. Cleaved caspase-3 and PARP by Western blot and a DNA fragment assay by Cell Death Detection ELISA^PLUS kit were used for the detection of apoptosis. The DNA fragment assay was performed in three independent experiments, and P values were calculated by a Student's t-test. D: Suppression of ΔNp63 or total p63 induced cellular senescence. A253 cells transfected with a negative control, p63, ΔNp63, or TAp63 siRNA were assayed for senescence-associated β-galactosidase activity. Knockdown of total p63 induced p21, but not p16, expression.