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Dysregulated WNT/β-catenin signaling in murine testes results in a phenotype with complete germ cell loss that resembles human Sertoli cell-only syndrome. In other systems, including the ovary, dysregulated WNT/β-catenin induces tumorigenesis but no tumors are observed in the mutant testes without deletion of a tumor suppressor, such as phosphatase and tensin homolog (PTEN). Müllerian inhibiting substance (MIS, also known as AMH), a member of the transforming growth factor-β family of growth factors responsible for Müllerian duct regression in fetal males, has been shown to inhibit tumor growth in vitro and in vivo but its role as an endogenous tumor suppressor has never been reported. We have deleted the MIS type 2 receptor (MISR2), and thus MIS signaling, in mice with dysregulated WNT/β-catenin and show that these mice develop testicular stromal tumors with 100% penetrance within a few months postnatal. The tumors are highly proliferative and have characteristics of either Sertoli cell tumors or progenitor Leydig cell tumors based on their marker profiles and histology. Phosphorylated Sma and mothers against decapentaplegic-related homolog 1/5/8 is absent in the tumors and β-catenin target genes are induced. The tumor suppressor TP53 is also highly expressed in the tumors, as is phosphorylated γH2AX, which is indicative of DNA damage. The phenotype of these tumors closely resembles those observed when PTEN is also deleted in mice with dysregulated WNT/β-catenin. Tumorigenesis in these mice provides conclusive evidence that physiological MIS signaling is a tumor suppressor mechanism and suggests that targeted treatment of MISR2-expressing cancers with therapeutic MIS should have a beneficial effect on tumor progression.

Mutations in the transforming growth factor beta type II receptor (TGFbetaRII) gene have been detected in several human cancer types exhibiting microsatellite instability. Using intron primers previously reported for examination of the entire coding region of the TGFbetaRII gene, 29 sporadic gastric cancers were screened with non-radioactive single strand conformation polymorphism and subsequent DNA sequencing analysis. Mutations of the TGFbetaRII gene were detected in three out of 29 tumors (10%). Two cases showed deletions in a polyadenine tract in both alleles and was positively associated with replication error. One case had an insertion of GA dinucleotide sequence in one allele. Mutations of the TGFbetaRII gene were restricted to exon 3 and other coding regions were not affected. Loss of heterozygosity was detected by analyzing a polymorphic site in intron 2. Three out of nine (33%) informative cases, which were all of intestinal type and advanced cases, showed loss of heterozygosity but neither TGFbetaRII mutation nor replication error was found in these cases. Immunoreactivity of TGFbetaRII in tumor tissues was reduced to a different extent in the gastric cancer with genetically abnormal transforming growth factor. Although the numbers studied are small, homozygous (A)10 deletion or loss of heterozygosity of TGFbetaRII is involved in tumorigenesis and progression of at least some part of sporadic gastric cancer.

The recent identification and cloning of mammalian transforming growth factor beta (TGFbeta) receptors permits further analysis of the importance of the TGFbeta family in intestinal biology. Expression of the type II TGFbeta receptor was examined in gastrointestinal cell lines and tissues. The 5.5 kb type II mRNA species was detected in poly-(A) mRNA isolated from the rat small bowel and colon. Northern blot analysis of RNA isolated from epithelial and non-epithelial small intestinal cell fractions showed the majority of receptor mRNA localized in the non-epithelial compartment. Immunohistochemical localization in the small intestine and colon supported the RNA findings; that is, expression was greatest in the lamina propria and muscularis. Staining was also detectable in the epithelium, where it was most prominent in the villus tip cells and absent in crypt cells. These findings mirror expression of TGFbeta in the epithelial compartment. The IEC-6, IPEC and RIE-1 cell lines, all of which are non-transformed, were growth inhibited by TGFbeta and expressed type II receptor mRNA and protein. By contrast, the ras-transfected RIE-1, HT-29, Caco-2 and SW-620 transformed lines were not growth inhibited by TGFbeta and all demonstrated a marked reduction in type II TGFbeta receptor mRNA expression and protein abundance by cross-linking. In conclusion, (i) colocalization of both ligand and receptor establishes the existence of potential autocrine and/or paracrine pathways for TGFbeta in the normal intestine and (ii) down-regulation of the type II TGFbeta receptor occurs in association with cellular transformation and may contribute to intestinal carcinogenesis.

Phenobarbital (PB) is a potent tumor promoter in rodent liver. In this study we investigated whether PB selectively promotes a population of initiated cells with reduced levels of transforming growth factor-beta (TGF beta) receptors types I, II and III. Liver tumors were induced in male Fischer F344 rats by diethylnitrosamine (DEN). Following induction the animals were divided into PB-treated (DEN/PB) and untreated groups (DEN). After 3 months of treatment half of the PB-treated rats were removed from PB for the final month (DEN/PB/OFF). At 4 months, the livers from rats in the three treatment groups were removed, tumors excised and frozen with matched surrounding normal liver tissue. The mRNA levels for the TGF beta receptors types I-III were significantly decreased in tumor tissue from DEN/PB rats when compared with surrounding normal liver tissue or tumors from age-matched untreated controls. In tumors from DEN/PB/OFF rats the TGF beta receptor types I-III were also significantly reduced compared with controls and not different to tumors from DEN/PB rats. There was no difference in the mRNA levels for the TGF beta receptors in tumors from rats exposed to DEN alone, when compared with the surrounding normal tissue. These results demonstrate that PB selectively promotes initiated cells with reduced levels of TGF beta types I-III receptors and suggests a mechanistic role for TGF beta in PB-induced liver tumor promotion.

Rat liver tumors initiated with N-nitrosodiethylamine (DEN) followed by promotion with phenobarbital (PB) were examined for expression of transforming growth factor-beta (TGF beta) type I, II and III receptors. RNase protection and TGF beta 1 affinity labeling assays were used to determine TGF beta receptor steady-state mRNA and protein levels, respectively. We have demonstrated that all three TGF beta receptors are expressed in both normal and malignant hepatic tissues. Long-term PB administration did not alter TGF beta receptor mRNA or protein levels in normal liver. However, type I, II and III TGF beta receptor mRNA and protein levels were decreased by approximately 50% in the DEN-initiated/PB-promoted liver tumors as compared to the receptor levels in normal liver tissue surrounding the tumors. In contrast, TGF beta receptor mRNA and protein levels were unchanged in liver tumors initiated with DEN but not PB-promoted. These data demonstrate that PB promotes the formation of a tumor phenotype that is characterized by a significantly reduced number of TGF beta type I, II and III receptors. This suggests that the down-regulation of TGF beta receptors in PB-promoted hepatic tumors may provide a selective growth advantage to the tumor cells by reducing the ability of TGF beta to inhibit their growth.