Inactivation of the Wwox gene accelerates forestomach tumor progression in vivo

Abstract

The WWOX gene encodes a tumor suppressor spanning the second most common human fragile site, FRA16D. Targeted deletion of the Wwox gene in mice led to an increased incidence of spontaneous and ethyl nitrosourea-induced tumors. In humans, loss of heterozygosity and reduced or loss of WWOX expression has been reported in esophageal squamous cell cancers (SCC). In the present study, we examined whether inactivation of the Wwox gene might lead to enhanced esophageal/forestomach tumorigenesis induced by N-nitrosomethylbenzylamine. Wwox+/- and Wwox+/+ mice were treated with six intragastric doses of N-nitrosomethylbenzylamine and observed for 15 subsequent weeks. Ninety-six percent (25 of 26) of Wwox+/- mice versus 29% (10 of 34) of Wwox+/+ mice developed forestomach tumors (P = 1.3 x 10(-7)). The number of tumors per forestomach was significantly greater in Wwox+/- than in Wwox+/+ mice (3.2 +/- 0.34 versus 0.47 +/- 0.17; P < 0.0001). In addition, 27% of Wwox+/- mice had invasive SCC in the forestomach, as compared with 0% of wild-type controls (P = 0.002). Intriguingly, forestomachs from Wwox+/- mice displayed moderately strong Wwox protein staining in the near-normal epithelium, but weak and diffuse staining in SCC in the same tissue section, a result suggesting that Wwox was haploinsufficient for the initiation of tumor development. Our findings provide the first in vivo evidence of the tumor suppressor function of WWOX in forestomach/esophageal carcinogenesis and suggest that inactivation of one allele of WWOX accelerates the predisposition of normal cells to malignant transformation.

Figure 1

Macroscopic appearance, histopathology, and expression of PCNA, KRT14, and Wwox protein in Wwox^+/+ and Wwox^+/- forestomachs at 15 wks after NMBA treatment. A, macroscopic appearance. Wwox^+/+ mouse no. 131 (a) and mouse no. 103 (b) showed a thickened forestomach and SCJ with small tumors. Wwox^+/- mouse no. 134 (c) displayed large fused tumors in the forestomach and SCJ, and mouse no. 102 had large fused tumors in the SCJ with tumors (>1 mm) in the forestomach (d). B, histopathology. H&E-stained sections of Wwox^+/+ forestomach from mouse no. 112 showed a thickened epithelium with basal cell hyperplasia (a); Wwox^+/- forestomach from mouse nos. 123 and 102 showed invasive SCC (d and g). PCNA-positive nuclei were mostly restricted to the basal cell layer in Wwox^+/+ forestomach epithelium (b), but was abundant in Wwox^+/- SCC mice nos. 123 and 102 (e and h; near serial sections of d and g). KRT14 expression showed basal cell staining in Wwox^+/+ forestomach from mouse no. 112 (c, near serial section of a), but intense and abundant staining in invasive SCC from Wwox^+/- forestomach from mouse nos. 123 and 102 (f and I, near serial sections of d and g). C, Wwox protein expression was strong in Wwox^+/+ forestomach epithelium (a). In Wwox^+/- forestomach from mouse no. 102, Wwox protein expression was moderately strong in areas of the epithelium showing mild hyperplasia (b), weak and diffuse in dysplasia (c), and very weak in SCC (d, near serial sections of B, g). Bars, 5 mm (A); 100 μm (B); and 25 μm (C).

Figure 2

Tumor multiplicity in Wwox^+/+ and Wwox^+/- forestomach. The number of forestomach tumors per mice was analyzed by one-way ANOVA (columns, mean; bars, SE). The number of small tumors (<1 mm) per mouse in Wwox^+/- mice was ~6-fold higher than in Wwox^+/+ mice (*, P < 0.0001). The number of large tumors (≥1 mm) per mouse was ~8-fold higher in Wwox^+/- mice than in wild-type littermates (**, P < 0.0001). All statistical tests were two-sided.