Role of beta-catenin/T-cell factor-regulated genes in ovarian endometrioid adenocarcinomas

Abstract

In various cancers, inactivating mutations in the adenomatous polyposis coli or Axin tumor suppressor proteins or activating mutations in beta-catenin's amino-terminal domain elevate beta-catenin levels, resulting in marked effects on T-cell factor (TCF)-regulated transcription. Several candidate beta-catenin/TCF-regulated genes in cancer have been proposed. Expression of a few of these genes has been studied in primary human cancers, but most studies have focused on colon cancers and not on other cancer types that harbor mutational defects in adenomatous polyposis coli, AXIN, or beta-catenin. Mutations leading to beta-catenin deregulation are found in nearly half of ovarian endometrioid adenocarcinomas (OEAs). We report here on the expression of 6 candidate beta-catenin/TCF-regulated genes in a panel of 44 primary OEAs, more than a third of which carry demonstrable defects in beta-catenin regulation. Using quantitative assays of gene expression, we found significantly elevated expression of the MMP-7, CCND1 (Cyclin D1), CX43 (Connexin 43), PPAR-delta, and ITF2 genes in OEAs with deregulated beta-catenin. This correlation was not observed for c-myc, another putative beta-catenin/TCF-regulated gene. Immunohistochemical studies confirmed that overexpression of cyclin D1 and MMP-7 was highly associated with nuclear accumulation of beta-catenin and mutational defects of the Wnt/beta-catenin/TCF-signaling pathway. Our findings indicate cyclin D1, MMP-7, connexin 43, PPAR-delta, and ITF-2, likely play important roles in the pathogenesis of those OEAs that manifest defects in beta-catenin regulation.

Figure 1.

Ribonuclease (RNase) protection assay of candidate β-catenin/Tcf target genes. Representative data from 22 tumors (11 tumors with altered β-catenin levels and localization and documented CTNNB1 or APC gene mutations and 11 tumors with intact β-catenin regulation) are shown. Five μg of total RNA from each tumor sample was hybridized to anti-sense ³²P-labeled riboprobes for c-myc (320 bp), CCND1 (286 bp), PPAR-δ (255 bp), MMP-7 (226 bp), ITF2 (203 bp), CX43 (180 bp), and L32 (76 bp). After overnight hybridization, samples were treated with RNases. Protected probes were extracted, precipitated, and separated by electrophoresis on 6% polyacrylamide denaturing gels. The intensity of the protected fragments in each tumor sample was quantitated by PhosphorImager analysis and compared to the intensity of the L32-specific fragment.

Figure 2.

Average expression of candidate β-catenin/TCF target genes in OEAs with β-catenin regulation defects or intact β-catenin regulation. The data in Table 1 ▶ were used to determine the average expression of the indicated candidate genes across groups of tumors with and without known or presumptive Wnt pathway defects. The normalized data (normalized to L32 expression) were analyzed using a two-tailed Student’s t-test assuming equal variance. All genes except c-myc showed significantly increased expression in the OEAs with defective β-catenin regulation.

Figure 3.

Correlation of β-catenin staining with staining for cyclin D1 and MMP-7 in representative OEAs. Serial sections of an OEA with mutant β-catenin (OE-47T) showed nuclear staining of β-catenin (A) and nuclear staining for cyclin D1 (B). A tumor with biallelic inactivation of APC (OE-32T) also showed nuclear β-catenin localization (C) and positive staining for cyclin D1 (D) in the neoplastic cells. In contrast, a tumor lacking β-catenin mutation (OE-20T) showed a membranous pattern of β-catenin staining (E) and lack of definitive immunoreactivity for cyclin D1 (F). Serial sections of an OEA with mutant β-catenin (OE-18T) and nuclear β-catenin staining (G) showed strong immunoreactivity for MMP-7 (H). A tumor with wild-type β-catenin and membranous β-catenin immunoreactivity (I) showed no staining for MMP-7 (J) (OE-23T). Original magnifications, ×400.